Formulation for anti-alpha4beta7 antibody

ABSTRACT

Antibody formulations are described comprising a mixture of an anti-α4β7 antibody, an antioxidant or chelator, and at least one free amino acid. The disclosed formulations may have improved stability, reduced aggregate formation, or both. The present invention further provides a safe dosing regimen of these antibody formulations that is easy to follow, and which results in a therapeutically effective amount of the anti-α4β7 antibody in vivo.

RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 15/983,791 filed on May 18, 2018, which is a continuation ofU.S. Non-Provisional application Ser. No. 14/114,835 filed on Feb. 24,2014, now U.S. Pat. No. 10,040,855 issued on Aug. 7, 2018, which is theU.S. National Stage application of International Application No.PCT/US2012/036069 filed on May 2, 2012, which claims priority to U.S.Provisional Application 61/544,054 filed on Oct. 6, 2011 and U.S.Provisional Application 61/481,522 filed on May 2, 2011. The entirecontents of the foregoing applications are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 10, 2020, isnamed Sequence_Listing_T103022 1020 US.C3.txt and is 17,130 bytes insize.

BACKGROUND OF THE INVENTION

Advances in biotechnology have made it possible to produce a variety ofproteins for pharmaceutical applications using recombinant DNAtechniques. Because proteins are larger and more complex thantraditional organic and inorganic drugs (i.e., possessing multiplefunctional groups in addition to complex three-dimensional structures),the formulation of such proteins poses special problems. For a proteinto remain biologically active, a formulation must preserve theconformational integrity of at least a core sequence of the protein'samino acids, while at the same time protecting the protein's multiplefunctional groups from degradation. Proteins may suffer from a lack ofstability, and monoclonal and polyclonal antibodies in particular may berelatively unstable (See e.g., Wang, et al., J. Pharm Sci. 96:1-26(2007)). A large number of formulation options are available, and notone approach or system is suitable for all proteins. Several factors tobe considered have been reported (See e.g., Wang et al.).

Numerous characteristics may affect a protein's stability. In fact, evenin the case of purified antibodies, the antibody structures may beheterogenous, which further complicates the formulation of such systems.Moreover, the excipients included in antibody formulations preferablyminimize any potential immune response.

In the case of antibodies, preservation of the conformational integrityis even more important. Degradation pathways for proteins can involvechemical instability (i.e., any process which involves modification ofthe protein by bond formation or cleavage resulting in a new chemicalentity) or physical instability (i.e., changes in the higher orderstructure of the protein). Chemical instability is manifested in, forexample, deamidation, isomerization, hydrolysis, oxidation,fragmentation, glycan beta elimination or disulfide exchange. Physicalinstability can result from denaturation, aggregation, precipitation oradsorption, for example. The four most common protein degradationpathways are protein fragmentation, aggregation, deamidation, andoxidation. Consequences of chemical or physical instability oftherapeutic protein include a lowering of the effective administereddose, decreased safety of the therapy due to, for example irritation orimmunological reactivity, and more frequent manufacturing due to shortshelf life.

Several publications have disclosed generally various methods oftreating inflammatory bowel diseases, and provided dosing schemes foradministration of agents designed to treat inflammatory bowel disease.For example, WO 96/24673 discloses mucosal vascular addressins andtreatment of diseases associated with leukocyte recruitment to thegastrointestinal tract as a result of leukocyte binding to cellsexpressing MAdCAM. U.S. 2005/0095238 describes methods of treating adisease associated with leukocyte infiltration of mucosal tissue andadministration to a human an effective amount of a human or humanizedimmunoglobulin or antigen binding fragment having binding specificityfor α4β7 integrin. U.S. 2005/0095238 further describes various doses(e.g. 0.15, about 0.5, about 1.0, about 1.5 or about 2.0 mgimmunoglobulin or fragment per kg body weight) and various intervalsbetween doses (7, 14, 21, 28, or 30 days). However, the aforementionedpatents and publications do not disclose specific formulations of theanti-α4β7 antibody or the specific doses and dose regimens described andclaimed herein. Importantly, the aforementioned patents do not discloseformulations, doses, and dose regimens that provide for the methods oftreatment (supported by clinical trial data) described and claimedherein.

The antibody formulations of the present invention may be useful forinhibiting leukocyte binding to cells expressing MAdCAM and thereforeaid in treatment of inflammatory bowel diseases in patients. There is,accordingly, an urgent need to discover suitable dosages and dosingschedules of these compounds, and to develop formulations, preferablysubcutaneous formulations, which give rise to steady, therapeuticallyeffective blood levels of the antibody formulations over an extendedperiod of time in a stable and convenient form.

SUMMARY OF THE INVENTION

The invention relates to the identification of an antioxidant orchelator, and at least one amino acid, as useful excipients forformulating anti-α4β7 antibody formulations whose instability makes themsusceptible to deamidation, oxidation, isomerization and/or aggregation.The formulation improves stability, reduces aggregate formation andretards degradation of the antibody therein.

Thus, in a first aspect, the invention relates to a stable liquidpharmaceutical formulation comprising a mixture of an anti-α4β7antibody, an antioxidant or chelator and at least one free amino acid.

In some embodiments, the stable liquid pharmaceutical formulation hasless than about 1.0% aggregate formation after 12 months at roomtemperature. The stable liquid pharmaceutical formulation can have lessthan about 0.2% aggregate formation after 12 months at room temperature.

In some embodiments, the antioxidant or chelator is citrate. In someembodiments the chelator is EDTA.

In some embodiments, the free amino acid of the formulation ishistidine, alanine, arginine, glycine, glutamic acid, or any combinationthereof. The formulation can comprise between about 50 mM to about 175mM of free amino acid. The formulation can comprise between about 100 mMand about 175 mM of free amino acid. The ratio of free amino acid toantibody molar ratio can be at least 250:1.

The formulation can also contain a surfactant. The surfactant can bepolysorbate 20, polysorbate 80, a poloxamer, or any combination thereof.

In some embodiments, the molar ratio of the antioxidant to thesurfactant is about 3:1 to about 156:1.

The formulation can have a pH between about 6.3 and about 7.0. The pH ofthe formulation can be between about 6.5 and about 6.8. The formulationcan have a pH between about 6.1 and about 7.0, or between about 6.2 and6.8.

In some embodiments, the stable liquid pharmaceutical formulationcontains at least about 60 mg/ml to about 160 mg/ml anti-α4β7 antibody.The formulation can contain at least about 160 mg/ml anti-α4β7 antibody.The formulation can contain about 150 to about 180 mg/ml antibody orabout 165 mg/ml antibody.

In another aspect, the invention relates to a stable liquidpharmaceutical formulation comprising at least about 60 mg/ml to about160 mg/ml anti-α4β7 antibody, a buffering agent and at least about 10 mMcitrate. The buffering agent can be a histidine buffer.

In another aspect, the invention relates to a stable liquidpharmaceutical formulation comprising at least about 60 mg/ml to about180 mg/ml anti-α4β7 antibody, a buffering agent and at least about 5 mMcitrate. The buffering agent can be a histidine buffer.

In another aspect, the invention relates to a stable liquidpharmaceutical formulation comprising at least about 160 mg/ml anti-α4β7antibody and at least about 10 mM citrate. The formulation can furthercontain polysorbate 80.

In another aspect, the invention relates to a stable liquidpharmaceutical formulation comprising about 160 mg/ml anti-α4β7 antibodyand at least about 5 mM citrate. The formulation can further containpolysorbate 80.

In another aspect, the invention relates to a stable liquidpharmaceutical formulation comprising a mixture of anti-α4β7 antibody,citrate, histidine, arginine and polysorbate 80. The formulation can bepresent in a container, such as a vial, cartridge, syringe orautoinjector.

The anti-α4β7 antibody in the stable liquid pharmaceutical formulationof the invention can be vedolizumab. The formulation of the inventioncan be for subcutaneous, intravenous, or intramuscular administration.

In some aspects, the formulation can minimize immunogenicity of theanti-α4β7 antibody.

In another aspect, the invention relates to a method of treatinginflammatory bowel disease, comprising administering to a patient inneed thereof the stable liquid pharmaceutical formulation describedherein. The administering can be subcutaneous administering. Theadministering can be self-administering.

In yet another aspect, the invention relates to an article ofmanufacture, comprising a container, a stable liquid pharmaceuticalformulation described herein, and instructions for its use.

In one aspect, the invention relates to a method for treating a humanpatient suffering from inflammatory bowel disease, wherein the methodcomprises the step of administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment thereof is administered to the patient according to thefollowing dosing regimen: (a) initial doses, e.g., in an induction phasetreatment regimen, of 165 mg of the humanized immunoglobulin orantigen-binding fragment thereof as a subcutaneous injection every otherday for six doses; (b) followed at week six by a seventh and subsequentdoses, e.g., in a maintenance phase treatment regimen, of 165 mg of thehumanized immunoglobulin or antigen-binding fragment thereof as asubcutaneous injection every two weeks or every four weeks as needed;wherein the dosing regimen induces a clinical response and clinicalremission in the inflammatory bowel disease of the patient; and furtherwherein the humanized immunoglobulin or antigen-binding fragment hasbinding specificity for the α4β7 complex, wherein the antigen-bindingregion comprises three complementarity determining regions (CDR1, CDR2,and CDR3) of a light chain variable region and three complementaritydetermining regions (CDR1, CDR2, and CDR3) of a heavy chain variableregion of the amino acid sequence set forth below: light chain: CDR1 SEQID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

In one aspect, the invention relates to a method for treating a humanpatient suffering from inflammatory bowel disease, wherein the methodcomprises the step of administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to the following dosing regimen comprising aninduction phase of intravenous doses and a maintenance phase ofsubcutaneous doses: (a) an initial intravenous dose of 300 mg of thehumanized immunoglobulin or antigen-binding fragment thereof as anintravenous infusion; (b) followed by a second intravenous subsequentdose of 300 mg of the humanized immunoglobulin or antigen-bindingfragment thereof as an intravenous infusion at about two weeks after theinitial dose; (c) followed beginning at week six by a third andsubsequent doses of 165 mg of the humanized immunoglobulin orantigen-binding fragment thereof as a subcutaneous injection every week,every two weeks, every three weeks or every four weeks as needed;wherein the dosing regimen induces a clinical response and clinicalremission in the inflammatory bowel disease of the patient; and furtherwherein the humanized immunoglobulin or antigen-binding fragment hasbinding specificity for the α4β7 complex, wherein the antigen-bindingregion comprises three complementarity determining regions (CDR1, CDR2,and CDR3) of a light chain variable region and three complementaritydetermining regions (CDR1, CDR2, and CDR3) of a heavy chain variableregion of the amino acid sequence set forth below: light chain: CDR1 SEQID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

In another aspect, the invention relates to a dosing regimen for thetherapeutic treatment of inflammatory bowel disease, wherein the dosingregimen comprises the step of: administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to a subcutaneous or intramuscular dosing regimenwhich maintains a mean steady state trough serum concentration of theimmunoglobulin or antigen-binding fragment thereof of about 9 to about13 μg/mL; wherein the dosing regimen induces a clinical response andclinical remission in the inflammatory bowel disease of the patient; andfurther wherein the humanized immunoglobulin or antigen-binding fragmenthas binding specificity for the α4β7 complex, wherein theantigen-binding region comprises three complementarity determiningregions (CDR1, CDR2, and CDR3) of a light chain variable region andthree complementarity determining regions (CDR1, CDR2, and CDR3) of aheavy chain variable region of the amino acid sequence set forth below:light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13;heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

In another aspect, the invention relates to a dosing regimen for thetherapeutic treatment of inflammatory bowel disease, wherein the dosingregimen comprises the step of: administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to a subcutaneous or intramuscular dosing regimenwhich maintains a mean steady state trough serum concentrations of thehumanized immunoglobulin or antigen-binding fragment thereof of about 35to about 40 μg/mL; wherein the dosing regimen induces a clinicalresponse and clinical remission in the inflammatory bowel disease of thepatient; and further wherein the humanized immunoglobulin orantigen-binding fragment has binding specificity for the α4β7 complex,wherein the antigen-binding region comprises three complementaritydetermining regions (CDR1, CDR2, and CDR3) of a light chain variableregion and three complementarity determining regions (CDR1, CDR2, andCDR3) of a heavy chain variable region of the amino acid sequence setforth below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3SEQ ID NO: 10.

In another aspect, the invention relates to a method of treating a humanpatient suffering from inflammatory bowel disease, wherein the methodcomprises the step of: administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to the following dosing regimen: (a) a pluralityof induction phase doses of the humanized immunoglobulin orantigen-binding fragment thereof sufficient to achieve a mean troughserum concentration of about 20 to about 30 μg/mL of the humanizedimmunoglobulin or antigen-binding fragment thereof by about six weeks ofinitial dosing; (b) followed by a plurality of maintenance phase dosesof the humanized immunoglobulin or antigen-binding fragment thereof asneeded to maintain a mean steady state trough serum concentration ofabout 9 to about 13 μg/mL or about 35 to 40 μg/mL of the immunoglobulinor antigen-binding fragment thereof; wherein the dosing regimen inducesa clinical response and clinical remission in the inflammatory boweldisease of the patient; and further wherein the humanized immunoglobulinor antigen-binding fragment has binding specificity for the α4β7complex, wherein the antigen-binding region comprises threecomplementarity determining regions (CDR1, CDR2, and CDR3) of a lightchain variable region and three complementarity determining regions(CDR1, CDR2, and CDR3) of a heavy chain variable region of the aminoacid sequence set forth below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQID NO: 9, CDR3 SEQ ID NO: 10.

In some aspects, the formulation, method of treatment, dose and/or doseregimen ensure minimal likelihood that a patient will develop antibodiesreactive to the anti-α4β7 antibody.

The patient may have had a lack of an adequate response with, loss ofresponse to, or was intolerant to treatment with at least one of animmunomodulator, a tumor necrosis factor-alpha (TNF-α) antagonist orcombinations thereof.

The inflammatory bowel disease can be Crohn's disease or ulcerativecolitis. The inflammatory bowel disease can be moderate to severelyactive ulcerative colitis.

The dosing regimen can result in mucosal healing in patients sufferingfrom moderate to severely active ulcerative colitis.

The patient may have previously received treatment with at least onecorticosteroid for the inflammatory bowel disease. The patient mayconcurrently receive treatment with at least one corticosteroid for theinflammatory bowel disease. The dosing regimen can result in areduction, elimination or reduction and elimination of corticosteroiduse by the patient.

In some aspects, the humanized immunoglobulin or antigen-bindingfragment thereof is administered in a final dosage form at aconcentration of between about 1.0 mg/ml to about 1.4 mg/ml. Thehumanized immunoglobulin or antigen-binding fragment thereof can beadministered in a final dosage form of about 1.2 mg/ml.

In some aspects, the humanized immunoglobulin or antigen-bindingfragment is administered in a final dosage form having an amount ofanti-α4β7 antibody between about 70 to about 250 mg, between about 90 toabout 200 mg, between about 150 to about 180 mg, or at least 160 mg.

In some aspects, the dosing regimen does not alter the ratio of CD4 toCD8 in cerebrospinal fluid of patients receiving said treatment.

The patient can be a person 65 years of age or older and does notrequire any adjustment of the dosing regimen.

In some aspects the method of treatment with the anti-α4β7 antibodyformulation, the dose, or the dose regimen can minimize immunogenicityof the anti-α4β7 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are illustrations of a nucleotide sequence (SEQ ID NO:1)encoding the heavy chain of a humanized anti-α4β7 immunoglobulin, andthe deduced amino acid sequence of the heavy chain (SEQ ID NO:2). Thenucleotide sequence contains cloning sites (lower case), Kozak sequence(upper case, nucleotides 18-23 of SEQ ID NO:1) and leader sequence(lower case, nucleotides 24-86 of SEQ ID NO:1) at the 5′ end of theheavy chain. The open reading frame of the nucleotide sequence isnucleotides 24-1433 of SEQ ID NO:1.

FIG. 2 is an illustration of a nucleotide sequence (SEQ ID NO:3)encoding the light chain of a humanized immunoglobulin referred toherein as vedolizumab, and the deduced amino acid sequence (SEQ ID NO:4) of the light chain. The nucleotide sequence contains cloning sites(lower case), Kozak sequence (upper case, nucleotides 18-23 of SEQ IDNO:3) and leader sequence (lower case, nucleotides 24-80 of SEQ ID NO:3)at the 5′ end of the heavy chain. The open reading frame of thenucleotide sequence is nucleotides 24-737 of SEQ ID NO:3.

FIG. 3 is an alignment of the amino acid sequences of (A) the maturehumanized light chain (amino acids 20-238 of SEQ ID NO:4) of thehumanized immunoglobulin referred to herein as vedolizumab and (B) themature humanized light chain of the humanized immunoglobulin referred toherein as LDP-02 (SEQ ID NO:5). (Regarding LDP-02, see, WO 98/06248 andFeagan et al., N. Eng. J. Med. 352:2499-2507 (2005)). Feagan et al.describe a clinical study of LDP-02, but in the article they refer toLDP-02 as MLN02.) The alignment illustrates that the amino acidsequences of the light chains of vedolizumab and LDP-02 differ atpositions 114 and 115 of the mature light chains.

FIG. 4 is an alignment of amino acid sequences of (A) a generic humankappa light chain constant region (SEQ ID NO:6) and (B) a generic murinekappa light chain constant region (SEQ ID NO:7). The amino acid residuesThr and Val (which are present at positions 114 and 115 of the maturevedolizumab light chain (amino acids 133 and 134 of SEQ ID NO:4)) arepresent in the constant region of the human kappa light chain, whereasthe amino acid residues Ala and Asp (which are present at positions 114and 115 of the mature LDP-02 light chain (SEQ ID NO:5)) are present inthe constant region of the mouse kappa light chain.

FIG. 5 is a map of vector pLKTOK38D (also referred to aspTOK38MLN02-TV), which encodes the humanized heavy chain and thehumanized light chain of MLN02, and is suitable for producingvedolizumab in CHO cells. (See, U.S. Patent Application Publication No.2004/0033561 A1 which discloses pLKTOK38. pLKTOK38D is a variant ofpLKTOK38 in which the restriction sites indicated on the map flank thesequence encoding the light chain variable region.)

FIG. 6 shows the SEC aggregates slope of formation (% per day) as aresult of changes to protein concentration, pH and surfactant:proteinmolar ratio. At a pH range of 6.0 to 6.5, the formation of aggregateswas similar for formulation with the polysorbate 80:protein molar ratiorange of 0.7 to 1.5.

FIG. 7 is a graph showing that at polysorbate 80:protein molar ratiosgreater than 1.5, the aggregate formation rate increases as pHincreases.

FIG. 8 is a graph showing the effect of excipients on the formation ofaggregates. 25 mM citrate, 5 mM citrate, 5 mM EDTA, 25 mM cysteine, or 5mM cysteine was added to formulations. All three excipients reduced theformation of aggregates.

FIG. 9 is a set of graphs that shows reduction in aggregate formationwith the presence of 25 mM citrate in the formulation, and a correlationbetween increased protein concentration and increased rate of aggregateformation.

FIG. 10 is a graph showing the results of the CEX species degradation at40° C. The data shows the influence of pH change on CEX degradation.

FIG. 11 is a graph showing the effect of temperature on the pH offormulations. The pH of formulations containing histidine decrease withtemperature, whereas the pH of citrate formulations is not affected bytemperature.

FIG. 12 is a graph showing the percentage of CEX major isoform over aperiod of twelve months. Formulations having a pH of 6.0-6.2 showedabout 1-2% fewer major isoform than formulations having a pH of 6.3-6.4.

FIG. 13 shows a set of graphs that demonstrate that viscosity isaffected mainly by protein concentration and pH. Sucrose, histidine andarginine additions are shown to have a minor affect on the viscosity ofthe formulation.

FIGS. 14A and 14B show the amino acid sequences of (A) the mature humanGM607′CL antibody kappa light chain variable region and (B) the human21/28′CL heavy chain variable region.

FIG. 15 shows components of a protein product in a pre-filled syringe.

FIGS. 16A and 16B show the effect of (A) protein concentration and (B)viscosity on the injection force of various syringes tested.

FIG. 17A shows the initial glide force as a function of proteinconcentration and the needle size. FIG. 17B shows the initial glideforce for each syringe manufacturer and needle size.

FIG. 18 shows the absorption profile of vedolizumab. The graph showsthat concentrations of the intramuscular and subcutaneous dosesgenerally overlap. There are no apparent gross differences in theabsorption profiles of these routes of administration.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a pharmaceutical formulation comprisinganti-α4β7 antibodies. The pharmaceutical formulation may be a mixturecomprising an antioxidant or chelator (e.g., citrate), anti-α4β7antibody and a free amino acid. The pharmaceutical formulation may be ina solid or liquid form.

Definitions

The term “pharmaceutical formulation” refers to a preparation thatcontains an anti-α4β7 antibody in such form as to permit the biologicalactivity of the antibody to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “stable” formulation is one in which the antibody thereinsubstantially retains its physical stability and/or its chemicalstability and/or its biological activity upon storage. In one aspect,the formulation substantially retains its physical and chemicalstability, as well as its biological activity upon storage. The storageperiod is generally selected based on the intended shelf-life of theformulation. Various analytical techniques for measuring proteinstability are available in the art and are reviewed, for example, inPeptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., MarcelDekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. DrugDelivery Rev. 10: 29-90 (1993). Stability can be measured at a selectedtemperature for a selected time period. For example, the liquidformulation is stable at about 40° C. for at least about 3 days, 5 days,1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks. In anotheraspect, the lyophilized formulation is stable at about 40° C. for atleast about 2-4 weeks, at least about 3 months, at least about 6 months,at least about 9 months, at least about 12 months, or at least about 18months. The liquid and/or lyophilized formulation in another aspect isstable at about 5° C. and/or 25° C. for at least about 1 month, at leastabout 3 months, at least about 6 months, at least about 9 months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, or at least about 36 months; and/orstable at about −20° C. and/or −70° C. for at least about 1 month, atleast about 3 months, at least about 6 months, at least about 9 months,at least about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months. Furthermore, the liquidformulation may, in some embodiments, be stable following freezing (to,e.g., −80° C.) and thawing, for example following 1, 2 or 3 cycles offreezing and thawing.

The stability of a liquid formulation can be evaluated qualitativelyand/or quantitatively in a variety of different ways, includingevaluation of dimer, multimer and/or aggregate formation (for exampleusing size exclusion chromatography (SEC), matrix-assisted laserdesorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS),analytical ultracentrifugation, light scattering (photon correlationspectroscopy, dynamic light scattering (DLS), static light scattering,multi-angle laser light scattering (MALLS)), flow-based microscopicimaging, electronic impedance (coulter) counting, light obscuration orother liquid particle counting system, by measuring turbidity, and/or byvisual inspection); by assessing charge heterogeneity using cationexchange chromatography (CEX), isoelectric focusing (IEF), e.g.capillary technique (cIEF), or capillary zone electrophoresis;amino-terminal or carboxy-terminal sequence analysis; mass spectrometricanalysis; SDS-PAGE or SEC analysis to compare fragmented, intact andmultimeric (i.e., dimeric, trimeric, etc.) antibody; peptide map (forexample tryptic or LYS-C) analysis; evaluating biological activity orantigen binding function of the antibody; and the like. Stability of asolid-state formulation can also be evaluated qualitatively and/orquantitatively in a variety of different ways, including direct tests,such as identifying crystal structure by X-Ray Powder Diffraction(XRPD); evaluating antibody structure in the solid state using FourierTransform Infrared Spectroscopy (FTIR); and measuring thermaltransitions in the lyophilized solid (melting, glass transition, etc.)using Differential Scanning calorimetry (DSC) and indirect tests such asmeasuring moisture content by Karl Fisher test, e.g., to extrapolate thelikelihood of chemical instability through hydrolysis. Instability mayinvolve any one or more of: aggregation (e.g., non-covalent solubleaggregation, covalent soluble aggregation (e.g., disulfide bondrearrangement/scrambling), insoluble aggregation), deamidation (e.g. Asndeamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Aspisomeriation), clipping/hydrolysis/fragmentation (e.g. hinge regionfragmentation), succinimide formation, N-terminal extension, C-terminalprocessing, glycosylation differences, and the like.

A “deamidated” monoclonal antibody is one in which one or moreasparagine or glutamine residue thereof has been derivatized, e.g. to anaspartic acid or an iso-aspartic acid.

An antibody which is “susceptible to deamidation” is one comprising oneor more residue which has been found to be prone to deamidate.

An antibody which is “susceptible to oxidation” is an antibodycomprising one or more residue which has been found to be prone tooxidation.

An antibody which is “susceptible to aggregation” is one which has beenfound to aggregate with other antibody molecule(s), especially uponfreezing, heating, drying, reconstituting and/or agitation.

An antibody which is “susceptible to fragmentation” is one which hasbeen found to be cleaved into two or more fragments, for example at ahinge region thereof.

By “reducing deamidation, oxidation, aggregation, or fragmentation” isintended to mean preventing or decreasing (e.g., to 80%, 60%, 50%, 40%,30%, 20% or 10% of) the amount of deamidation, aggregation, orfragmentation relative to the monoclonal antibody formulated at adifferent pH or in a different buffer.

An “aggregate”, “SEC aggregate”, or “soluble aggregate” is more than oneand less than or equal to ten antibody proteins and/or fragmentsassociated together through covalent, ionic, or hydrophobic interactionsto form a larger protein body.

An “insoluble aggregate” or “particle” is greater than ten antibodyproteins and/or fragments associated together through covalent, ionic,or hydrophobic interactions to form a larger protein body.

As used herein, “biological activity” of a monoclonal antibody refers tothe ability of the antibody to bind to antigen and result in ameasurable biological response which can be measured in vitro or invivo. Such activity may be antagonistic or agonistic.

The cell surface molecule, “α4β7 integrin,” or “α4β7,” is a heterodimerof an α₄ chain (CD49D, ITGA4) and a β₇ chain (ITGB7). Each chain canform a heterodimer with an alternative integrin chain, to form forexample α₄β₁ or α_(E)β₇. Human α₄ and β₇ genes (GenBank (National Centerfor Biotechnology Information, Bethesda, Md.) RefSeq Accession numbersNM_000885 and NM_000889, respectively) are expressed by B and Tlymphocytes, particularly memory CD4+ lymphocytes. Typical of manyintegrins, α4β7 can exist in either a resting or activated state.Ligands for α4β7 include vascular cell adhesion molecule (VCAM),fibronectin and mucosal addressin (MAdCAM, e.g., MAdCAM-1).

As used herein, a human immunoglobulin or antigen-binding fragmentthereof that has “binding specificity for the α4β7 complex” binds toα4β7, but not to α4β1 or αEB7.

As used herein, an “isotonic” formulation has substantially the sameosmotic pressure as human blood. Isotonic formulations will generallyhave an osmotic pressure from about 250 to 350 mOsm. Isotonicity can bemeasured using a vapor pressure or ice-freezing type osmometer, forexample.

As used herein, “buffering agent” refers to a buffer that resistschanges in pH by the action of its acid-base conjugate components. Thebuffering agent may be present in a liquid or solid formulation of theinvention. In some embodiments, the buffering agent of this inventionadjusts the pH of the formulation to about 5.0 to about 7.5, to about pH5.5 to about 7.5, to about pH 6.0 to about 7.0, or to a pH of about 6.3to about 6.5. In one aspect, examples of buffering agents that alone orin combination, will control the pH in the 5.0 to 7.5 range includeacetate, succinate, gluconate, histidine, citrate, phosphate, maleate,cacodylate, 2-[N-morpholino]ethanesulfonic acid (MES),bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris),N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine and otherorganic acid buffers. In another aspect, the buffering agent herein ishistidine or citrate.

A “histidine buffer” is a buffer comprising histidine ions. Examples ofhistidine buffers include histidine chloride, histidine acetate,histidine phosphate, histidine sulfate solutions. The histidine bufferor histidine-HCl buffer has a pH between about pH 5.5 to about 7.0,between about pH 6.1 to about 6.9, or about pH 6.5.

A “citrate buffer” is a buffer comprising citrate ions. Examples ofcitrate buffers include sodium citrate, ammonium citrate, calciumcitrate, and potassium citrate solutions. The citrate buffer has a pH ofabout 3.0 to 6.2, about pH 5.5 to 6.5, about pH 6.1 to about 6.5, aboutpH 6.1, about pH 6.2, or about pH 6.5.

A “saccharide” herein is a compound that has a general formula(CH₂O)_(n) and derivatives thereof, including monosaccharides,disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducingsugars, nonreducing sugars, and the like. Examples of saccharides hereininclude glucose, sucrose, trehalose, lactose, fructose, maltose,dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol,mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose,lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, andthe like. A saccharide can be a lyoprotectant. In one aspect, asaccharide herein is a nonreducing disaccharide, such as sucrose.

Herein, a “surfactant” refers to an agent that lowers surface tension ofa liquid. In one aspect, the surfactant is a nonionic surfactant.Examples of surfactants herein include polysorbate (polyoxyethylenesorbitan monolaurate, for example, polysorbate 20 and polysorbate 80);TRITON (t-Octylphenoxypolyethoxyethanol, nonionic detergent, UnionCarbide subsidiary of Dow Chemical Co., Midland Mich.); sodium dodecylsulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; sorbitan monopalmitate; and the MONAQUAT series(Mona Industries, Inc., Paterson, N.J.); polyethyl glycol (PEG),polypropylene glycol (PPG), and copolymers of poloxyethylene andpoloxypropylene glycol (e.g. Pluronics/Poloxamer, PF68 etc); etc. Inanother aspect, the surfactant herein is polysorbate 80.

The term “chelator” refers to an agent that binds to an atom throughmore than one bond. In one aspect, examples of chelators herein includecitrate, ethylenediaminetetraacetic acid, ethyleneglycoltetraacetic acid(EGTA), dimercaprol, diethylenetriaminepentaacetic acid, andN,N-bis(carboxymethyl)glycine. In another aspect, the chelator iscitrate or EDTA.

The term “antioxidant” refers to an agent that inhibits the oxidation ofother molecules. Examples of antioxidants herein include citrate, lipoicacid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine,phosphonate compounds, e.g., etidronic acid, desferoxamine and malate.

The term “antibody” herein is used in the broadest sense andspecifically covers full length monoclonal antibodies, immunoglobulins,polyclonal antibodies, multispecific antibodies (e.g. bispecificantibodies) formed from at least two full length antibodies, e.g., eachto a different antigen or epitope, and individual antigen bindingfragments, including dAbs, scFv, Fab, F(ab)′₂, Fab′, including human,humanized and antibodies from non-human species and recombinant antigenbinding forms such as monobodies and diabodies.

Molar amounts and ratios of anti-α4β7 antibody to other excipientsdescribed herein are calculated on the assumption of an approximatemolecular weight of about 150,000 daltons for the antibody. The actualantibody molecular weight may differ from 150,000 daltons, depending onamino acid composition or post-translational modification, e.g., asdependent on the cell line used to express the antibody. Actual antibodymolecular weight can be +/−5% of 150,000 daltons.

The term “human antibody” includes an antibody that possesses a sequencethat is derived from a human germ-line immunoglobulin sequence, such asan antibody derived from transgenic mice having human immunoglobulingenes (e.g., XENOMOUSE genetically engineered mice (Abgenix, Fremont,Calif.), HUMAB-MOUSE®, KIRIN TC MOUSE™ transchromosome mice, KMMOUSE®(MEDAREX, Princeton, N.J.)), human phage display libraries, humanmyeloma cells, or human B cells.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen binding sequences derived from a non-humanprimate (e.g. Old World Monkey, Ape etc) and human constant regionsequences.

“Antigen binding fragments” of the humanized immunoglobulin prepared inthe formulation of the invention comprise at least the variable regionsof the heavy and/or light chains of an anti-α4β7 antibody. For example,an antigen binding fragment of vedolizumab comprises amino acid residues20-131 of the humanized light chain sequence of SEQ ID NO:4. Examples ofsuch antigen binding fragments include Fab fragments, Fab′ fragments,scFv and F(ab′)₂ fragments of a humanized immunoglobulin known in theart. Antigen binding fragments of the humanized immunoglobulin of theinvention can be produced by enzymatic cleavage or by recombinanttechniques. For instance, papain or pepsin cleavage can be used togenerate Fab or F(ab′)₂ fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons have been introduced upstream of the naturalstop site. For example, a recombinant construct encoding the heavy chainof an F(ab′)₂ fragment can be designed to include DNA sequences encodingthe CH_(I) domain and hinge region of the heavy chain. In one aspect,antigen binding fragments inhibit binding of α4β7 integrin to one ormore of its ligands (e.g. the mucosal addressin MAdCAM (e.g., MAdCAM-1),fibronectin).

Papain digestion of antibodies produces two identical antigen bindingfragments, called “Fab” fragments, each with a single antigen bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding sites and is still capable of cross-linkingantigen.

“Fv” is an antibody fragment which consists of a dimer of one heavychain variable domain and one light chain variable domain innon-covalent association.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In one aspect, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with two antigenbinding sites, which fragments comprise a variable heavy domain (V_(H))connected to a variable light domain (V_(L)) in the same polypeptidechain (V_(H)-V_(L)). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “full length antibody” is one which comprises an antigen bindingvariable region as well as a light chain constant domain (C_(L)) andheavy chain constant domains, C_(H1), C_(H2) and C_(H3). The constantdomains may be native sequence constant domains (e.g. human nativesequence constant domains) or amino acid sequence variants thereof. Inone aspect, the full length antibody has one or more effector functions.

An “amino acid sequence variant” antibody herein is an antibody with anamino acid sequence which differs from a main species antibody.Ordinarily, amino acid sequence variants will possess at least about70%, at least about 80%, at least about 85%, at least about 90%, or atleast about 95% homology with the main species antibody. The amino acidsequence variants possess substitutions, deletions, and/or additions atcertain positions within or adjacent to the amino acid sequence of themain species antibody, but retain antigen binding activity. Variationsin sequence of the constant regions of the antibody will have lesseffect on the antigen binding activity than variations in the variableregions. In the variable regions, amino acid sequence variants will beat least about 90% homologous, at least about 95% homologous, at leastabout 97% homologous, at least about 98% homologous, or at least about99% homologous with the main species antibody.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart.

A “therapeutic monoclonal antibody” is an antibody used for therapy of ahuman subject. Therapeutic monoclonal antibodies disclosed hereininclude anti-α4β7 antibodies.

A “glycosylation variant” antibody herein is an antibody with one ormore carbohydrate moeities attached thereto which differ from one ormore carbohydate moieties attached to a main species antibody. Examplesof glycosylation variants herein include antibody with a G1 or G2oligosaccharide structure, instead of a G0 oligosaccharide structure,attached to an Fc region thereof, antibody with one or two carbohydratemoieties attached to one or two light chains thereof, antibody with nocarbohydrate attached to one or two heavy chains of the antibody, etc,and combinations of glycosylation alterations.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), and the like.

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to different“classes”. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one aspect, the FcR is anative sequence human FcR. In another aspect, the FcR is one which bindsan IgG antibody (a gamma receptor) and includes receptors of the FcγRI,FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (See, review in M. Daeron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med./26:33-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined. The hypervariableregion or the CDRs thereof can be transferred from one antibody chain toanother or to another protein to confer antigen binding specificity tothe resulting (composite) antibody or binding protein.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable regions thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). In one aspect, affinitymatured antibodies will have nanomolar or even picomolar affinities forthe target antigen. Affinity matured antibodies are produced byprocedures known in the art. Marks et al. Bio/Technology 10:779-783(1992) describes affinity maturation by VH and VL domain shuffling.Random mutagenesis of CDR and/or framework residues is described by:Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier etal. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins etal., J. Mol. Biol. 226:889-896 (1992).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment. In certainembodiments, the antibody will be purified (1) to greater than 95% byweight of protein as determined by the Lowry method, and alternatively,more than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, isolated antibody will be preparedby at least one purification step.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disease as well as those in which the disease or its recurrenceis to be prevented. Hence, the patient to be treated herein may havebeen diagnosed as having the disease or may be predisposed orsusceptible to the disease. The terms “patient” and “subject” are usedinterchangeably herein.

The antibody which is formulated is substantially pure and desirablysubstantially homogeneous (i.e. free from contaminating proteins etc).“Substantially pure” antibody means a composition comprising at leastabout 90% antibody by weight, based on total weight of the protein,alternatively, at least about 95% or 97% by weight. “Substantiallyhomogeneous” antibody means a composition comprising protein wherein atleast about 99% by weight of protein is specific antibody, e.g.,anti-α4β7 antibody, based on total weight of the protein.

“Clinical remission” as used herein with reference to ulcerative colitissubjects refers to a complete Mayo score of 2 or less points and noindividual subscore greater than 1 point. Crohn's disease “clinicalremission” refers to a CDAI score of 150 points or less.

A “clinical response” as used herein with reference to ulcerativecolitis subjects refers to a reduction in complete Mayo score of 3 orgreater points and 30% from baseline, (or a partial Mayo score of 2 orgreater points and 25% or greater from baseline, if the complete Mayoscore was not performed at the visit) with an accompanying decrease inrectal bleeding subscore of 1 or greater points or absolute rectalbleeding score of 1 or less point. A “clinical response” as used hereinwith reference to Crohn's disease subjects refers to a 70 point orgreater decrease in CDAI score from baseline (week 0).

“Mucosal healing” as used herein with reference to ulcerative colitissubjects refers to an endoscopic subscore of 1 point or less.

As used herein, “treatment failure” refers to disease worsening, a needfor rescue medications or surgical intervention for treatment ofulcerative colitis or Crohn's disease. A rescue medication is any newmedication or any increase in dose of a baseline medication required totreat new or unresolved ulcerative colitis or Crohn's disease symptoms(other than antidiarrheals for control of chronic diarrhea).

Formulations

As described herein, it has been discovered that anti-α4β7 antibodiesare more stable when formulated with an antioxidant or chelator. Inaddition, as described herein, anti-α4β7 antibodies may be formulated toreduce aggregate formation (e.g., the amount of polysorbate 80 in theformulation may be reduced). For example, formulations that comprisecitrate or EDTA and anti-α4β7 antibodies decrease the rate of antibodyaggregate formation during storage. Formulations may also be storedwithout oxygen to reduce aggregate formation. In one embodiment, theformulation has an antibody aggregate formation of less than about 2.5%at 25° C. after 12 months. In one embodiment, the formulation has anantibody aggregate formation of less than about 2.0% at 25° C. after 12months. In one embodiment, the formulation has an antibody aggregateformation of less than about 1.6% at 25° C. after 12 months. In oneembodiment, the formulation has an antibody aggregate formation of lessthan about 1.3% at 25° C. after 12 months. In one embodiment, theformulation has an antibody aggregate formation of less than about 1.0%at 25° C. after 12 months. In another embodiment, the formulation has anantibody aggregate formulation of less than about 0.5% at 5° C. after 12months. In another embodiment, the formulation has an antibody aggregateformulation of less than about 0.3% at 5° C. after 12 months.

The present invention provides, in a first aspect, a stable anti-α4β7antibody formulation. The formulation comprises an anti-α4β7 antibodyand an antioxidant or chelator. The formulation also comprises abuffering agent that may be one or more free amino acids. Theformulation may optionally further comprise a surfactant. The antibodyin the formulation may be a full length antibody or an antigen bindingfragment thereof, such as a Fab, Fv, scFv, Fab′ or F(ab′)2 fragment.

Aggregate formation can be reduced by removing oxygen from theformulation. Alternatively, the formulation can contain an antioxidantor chelator. In one aspect, exemplary antioxidants and chelators thatcan be included in the formulation include lipoic acid, uric acid,glutathione, tocopherol, carotene, lycopene, cysteine,ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid(EGTA), dimercaprol, diethylenetriaminepentaacetic acid, andN,N-bis(carboxymethyl)glycine, phosphonate compounds, e.g., etidronicacid, desferoxamine, malate and citrate. Some antioxidants and chelatorscan decrease the rate of aggregate formation during storage of theformulation. In another aspect, the chelator and/or antioxidant iscitrate or EDTA. Exemplary chelator concentrations for liquidformulations are in the range of from about greater than 0 mM to about60 mM, about 5 mM to about 50 mM, about 5 mM to about 15 mM, about 10 mMto about 25 mM, and about 20 to about 30 mM. In another aspect, thechelator concentration is from about 0 mM to about 30 mM. In oneembodiment, the chelator and/or antioxidant is citrate, and the citrateconcentration is from about 0 mM to about 15 mM, about 0 mM to about 10mM, or about 0 mM to about 5 mM.

The formulation can contain any desired one free amino acid, which canbe in the L-form, the D-form or any desired mixture of these forms. Inone aspect, free amino acids that can be included in the formulationinclude, for example, histidine, alanine, arginine, glycine, glutamicacid, serine, lysine, tryptophan, valine, cysteine and combinationsthereof. Some amino acids can stabilize the proteins against degradationduring manufacturing, drying, lyophilization and/or storage, e.g.,through hydrogen bonds, salt bridges, antioxidant properties, orhydrophobic interactions or by exclusion from the protein surface Aminoacids can act as tonicity modifiers or can act to decrease viscosity ofthe formulation. In another aspect, free amino acids, such as histidineand arginine, can act as lyoprotectants, and do not crystallize whenlyophilized as components of the formulation. Free amino acids, such asglutamic acid and histidine, alone or in combination, can act asbuffering agents in aqueous solution in the pH range of 5 to 7.5. Instill yet another aspect, the formulation contains histidine, arginine,or a combination of histidine and arginine. In still yet another aspect,free amino acid concentrations for liquid formulations are in the rangefrom about 9 mM to about 0.5 M, for example, from about 10 mM to about90 mM, about 10 mM to about 75 mM, about 10 mM to about 40 mM, about 25mM to about 50 mM, about 15 mM to about 300 mM, about 20 mM to about 200mM, about 25 mM to about 150 mM, about 50 mM to about 75 mM, about 50 mMto about 120 mM, about 50 to about 150 mM, or about 50 mM or about 125mM.

The formulation can optionally further contain at least one surfactant,e.g., to control soluble and insoluble aggregate formation. In oneaspect, the surfactant is a non-ionic surfactant. In another aspect, thesurfactant is an ionic surfactant. Exemplary surfactants that can beincluded in the formulation include, for example, polysorbate 20,polysorbate 80, a poloxamer (Pluronic®) and combinations thereof. Whenpresent, the surfactant is generally included in an amount which reducesformation of insoluble aggregates of antibody, e.g., during bottling,freezing, drying, lyophilization and/or reconstitution, in the presenceof silicone, filling vials, prefilled syringes, and/or cartridges. Thesurfactant concentration is generally from about 0.0001% to about 1.0%,from about 0.01% to about 0.5%, for example, about 0.05%, 0.1%, 0.15%,0.20%, 0.3%, 0.4%, or 0.5% (w/v). Higher concentrations of surfactant,e.g., polysorbate 80 can lead to more SEC aggregate formation. Reducingthe concentration of polysorbate 80 can reduce SEC aggregate formationupon storage. In one aspect, the surfactant:antibody molar ratio is fromabout 0.7:1 to about 2.0:1. In another aspect, the surfactant: antibodymolar ratio is 1.5:1.

An embodiment of an anti-α4β7 antibody formulation contains a highconcentration of anti-α4β7 antibody. For example, in one embodiment, theliquid formulations can comprise at least about 60 mg/ml, at least about70 mg/ml, at least about 80 mg/ml, at least about 90 mg/ml, at leastabout 100 mg/ml, at least about 110 mg/ml, at least about 120 mg/ml, atleast about 130 mg/ml, at least about 140 mg/ml, at least about 150mg/ml, at least about 160 mg/ml, at least about 170 mg/ml, at leastabout 180 mg/ml, at least about 190 mg/ml, at least about 200 mg/ml, atleast about 250 mg/ml, at least about 300 mg/ml, from about 60 mg/ml toabout 190 mg/ml, from about 60 mg/ml to about 170 mg/ml anti-α4β7antibody, from about 150 mg/ml to about 180 mg/ml, or about 160 mg/ml orabout 165 mg/ml anti-α4β7 antibody. Alternatively, in another aspect,the liquid formulations can comprise at least about 154 mg/ml, at leastabout 176 mg/ml.

The formulation can be a liquid or a solid. Liquid formulations areaqueous solutions or suspensions, prepared in a suitable aqueoussolvent, such as water or an aqueous/organic mixture, such as wateralcohol mixtures. Liquid formulations have a pH between about 5.5 andabout 7.5, between about 6.0 and 7.3, between about 6.0 and about 7.0,between about 6.0 and 6.5, between about 6.0 and 6.3, between about 6.3and 7.1, or between about 6.4 and 7.0, or between 6.3 and 6.8, such asabout 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9. Liquidformulations can be kept at room temperature, refrigerated (e.g., 2-8°C.), or frozen (e.g., −20° C. or −70° C.) for storage.

A solid formulation can be prepared in any suitable way and can be inthe form of a cake or powder, for example, with the addition of alyoprotectant. In one aspect, the solid formulation is prepared bydrying a liquid formulation as described herein, for example bylyophilization or spray drying. When the formulation is a solidformulation, the formulation can have a moisture content of no more thanabout 5%, no more than about 4.5%, no more than about 4%, no more thanabout 3.5%, no more than about 3%, no more than about 2.5%, no more thanabout 2%, no more than about 1.5%, no more than about 1%, or issubstantially anhydrous. A solid formulation can be dissolved, i.e.reconstituted, in a suitable medium or solvent to become liquid suitablefor administration. Suitable solvents for reconstituting the solidformulation include water, isotonic saline, buffer, e.g.,phosphate-buffered saline, Ringer's (lactated or dextrose) solution,minimal essential medium, alcohol/aqueous solutions, dextrose solution,etc. The amount of solvent can result in a therapeutic proteinconcentration higher, the same, or lower than the concentration prior todrying. In another aspect, the reconstituted anti-α4β7 antibodyconcentration is the same concentration as in the pre-drying liquidformulation.

The formulation may be sterile, and this can be achieved according tothe procedures known to the skilled person for generating sterilepharmaceutical formulations suitable for administration to humansubjects, prior to, or following, preparation of the formulation. Theformulation can be sterilized as a liquid, e.g., before drying and/orafter reconstitution by filtration through small pores, through asepticprocessing or by exposure to ultraviolet radiation. Filter pore sizescan be 0.1 μm or 0.2 μm to filter microorganisms or 10 to 20 nm tofilter virus particles. Alternatively, or additionally, the driedformulation can be sterilized, e.g., by exposure to gamma radiation. Inone aspect, the anti-α4β7 antibody liquid formulation is sterilized byfiltration before drying.

In one aspect, the formulation is stable upon storage. Various stabilityassays are available to the skilled practitioner for confirming thestability of the formulation. For example, the antibody in the liquidformulation may be stable upon storage at about 25° C. for at leastabout 4 weeks, at least about 2 months, at least about 3 months, or atleast about 6 months, or at least about 9 months, or at least about 12months; at about 2-8° C. at least about 3 months, at least about 1 year,at least about 2 years, at least about 3 years or longer. Alternativelyor in addition, the antibody in the formulation may be stable uponstorage at about 15° C. for at least about 4 weeks, at least about 3months, at least about 6 months, at least about 9 months, at least about1 year, or longer. Alternatively or in addition, the antibody in theformulation may be stable upon storage at about −20° C. or −70° C. forat least about 4 weeks; at least about 3 months, at least about 6months, at least about 9 months, at least about 1 year, at least about 2years, at least about 3 years, at least about 4 years or longer.

Stability can be tested by evaluating physical stability, chemicalstability, and/or biological activity of the antibody in the formulationaround the time of formulation as well as following storage at the notedtemperatures. Physical and/or chemical stability of a liquid formulationor a reconstituted dry powder can be evaluated qualitatively and/orquantitatively in a variety of different ways (see, e.g., AnalyticalTechniques for Biopharmaceutical Development, Rodriguez-Diaz et al. eds.Informa Healthcare (2005)), including evaluation of soluble andinsoluble aggregate formation (for example using size exclusionchromatography, analytical ultracentrifugation, MALDI-TOF MS, lightscattering (dynamic (DLS) or MALLS), flow-based microscopic imaging, orother liquid particle counting system, by measuring turbidity, bydensity gradient centrifugation and/or by visual inspection); byassessing charge heterogeneity using cation exchange chromatography (seealso Vlasak and Ionescu, Curr. Pharm. Biotechnol. 9:468-481 (2008) andHarris et al. J. Chromatogr. B Biomed. Sci. Appl. 752:233-245 (2001)),isoelectric focusing or capillary zone electrophoresis; amino-terminalor carboxy terminal sequence analysis; mass spectrometric analysis;SDS-PAGE analysis to compare fragmented, intact and multimeric (i.e.,dimeric, trimeric, etc.) antibody; peptide map (for example tryptic orLYS- and the like). Instability may result in aggregation, deamidation(e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization(e.g., Asp isomerization), denaturation,clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation),succinimide formation, unpaired cysteine(s), N-terminal extension,C-terminal processing, glycosylation differences, etc. Biologicalactivity or antigen binding function, e.g., binding of the anti-α4β7antibody to MAdCAM (e.g., MAdCAM-1) or inhibition of the binding of acell expressing α4β7 integrin to MAdCAM (e.g., MAdCAM-1), e.g.,immobilized MAdCAM (e.g., MAdCAM-1), can be evaluated using varioustechniques available to the skilled practitioner (see e.g., Soler etal., J. Pharmacol. Exper. Ther. 330:864-875 (2009)). Measurement of themoisture content of a dry formulation can indicate how likely aformulation will undergo chemical or physical degradation, with highermoisture leading to more degradation.

A stable formulation can contribute to a low immunogenicity of ananti-α4β7 antibody. An immunogenic anti-α4β7 antibody can lead to ahuman-anti-human antibody (HAHA) response in human subjects or patients.Patients who develop a HAHA response to an anti-α4β7 antibody can haveadverse events (e.g., site infusion reaction) upon treatment or caneliminate anti-α4β7 antibody quickly, resulting in a lower dose thanplanned by treatment. A report (Feagen et al. (2005) N. Engl. J. Med.352:2499-2507) of early study of an anti-α4β7 antibody treatmentindicated that human antihuman antibodies developed by week 8 in 44% oftreated patients. The antibody in this study was stored as a liquid anddid not contain any polysorbate.

In some embodiments, the formulation can increase the proportion of HAHAnegative patients to at least 40%, at least 50%, at least 60%, at least70%, at least 80% or at least 90% of patients compared to the HAHAresults of a less stable formulation.

In some embodiments, an anti-α4β7 antibody formulation has ≥50% majorcharged isoform, ≥55% major charged isoform, or 65 to 70% major chargedisoform. In other aspects, a stable anti-α4β7 antibody formulation has≤45% acidic charged isoforms, ≤40% acidic charged isoforms, ≤30% acidiccharged isoforms or 22 to 28% acidic isoforms. In still other aspects, astable anti-α4β7 antibody formulation has ≤25% basic isoforms, ≤20%basic isoforms, ≤15% basic isoforms, about 5% basic isoforms or about10% basic isoforms. In one aspect, a stable anti-α4β7 antibodyformulation has ≥55% major isoform, ≤30% acidic isoforms and/or ≤20%basic isoforms, e.g., as determined by CEX. In another aspect, a stableanti-α4β7 antibody formulation has ≥50% major isoform, ≤45% acidicisoforms and/or <10% basic isoforms, e.g., as determined by cIEF.

In some aspects, an anti-α4β7 antibody dry, solid formulation has ≤10%moisture content, ≤5% moisture content or <2.5% moisture content. Thetime required for reconstitution is ≤60 minutes, ≤50 minutes or ≤40minutes or ≤30 minutes or ≤20 minutes.

Monomeric content and/or aggregate content (e.g., as dimers, trimers,tetramers, pentamers, oligomers and higher-order aggregates), i.e., inthe liquid formulation, or in the reconstituted formulation, can bemeasured by SEC, analytical ultracentrifugation, light scattering (DLSor MALLS), MALDI-TOF MS or nanoscale measurement, such as nanoparticletracking analysis NTA, NanoSight Ltd, Wiltshire, UK). Resolution,characterization and quantification of aggregate can be achieved in anumber of ways, including increasing the length of the SEC columnseparation, e.g., by a longer column or by serial attachment of a secondor more SEC column(s) in line with the initial analytical SEC column,supplementing SEC quantification of monomers with light scattering, orby using NTA.

In one embodiment, an anti-α4β7 antibody formulation has 90% monomericantibody, 95% monomeric antibody, or 97 to 99% monomeric antibody. Inanother embodiment, the majority of the material in an anti-α4β7antibody formulation has an average radius of ≤20 nm, ≤15 nm, ≤10 nm, orabout 5 to about 7 nm. In one aspect, an anti-α4β7 antibody formulationhas ≥80% amount heavy plus light chain by protein analysis. In oneaspect, there is ≥90% heavy plus light chain. In another aspect, ananti-α4β7 antibody formulation has ≤10% aggregate, ≤5% aggregate, ≤2.5%aggregate, ≤1.5% aggregate, ≤1.0% aggregate or ≤0.5% aggregate. Inanother aspect, stable anti-α4β7 antibody formulation has ≥96% monomerand/or ≤2.5% aggregate. In yet another aspect, a stable anti-α4β7antibody formulation has about 99% monomer and/or about <1% aggregate.

Particle sizes, greater than 1 to 2 micron, e.g., of aggregates orundissolved excipient, i.e., in the liquid formulation, or in thereconstituted formulation can be measured by light obscuration (e.g.,liquid particle counting system (HIAC) by Hach Ultra Analytics (GrantsPass, Oreg.)), microscopy, coulter counter, or digital (e.g.,flow-based) microscopic imaging based system such as microfluidicsimaging (MFI) by Brightwell (Ottawa, Calif.) or FLOWCAM® Image particleanalyzer by Fluid Imaging Technologies (Yarmouth, Me.). In one aspect,particle size in an anti-α4β7 antibody preparation is about 30 μm, about25 μm, about 10 μm, about 5 μm, about 2 μm or 1 μm or less. The amountof particles should be minimized in antibody formulations. In oneaspect, an amount of particles in an anti-α4β7 antibody formulation is<6000 particles ≥10 μm diameter and/or <600 particles ≥25 μm diameter inone dose (U.S. Pharmacopoeia Chp. 788, light obscuration countingmethod; half those amounts by microscopic quantification method). Inanother aspect, an amount of particles in a dose of an anti-α4β7antibody formulation is about 1000 particles ≥10 μm and about 0-100particles ≥25 μm (MFI method). In yet another aspect, an amount ofparticles per milliliter, e.g., by MFI measurement, in a dose of ananti-α4β7 antibody formulation is about 500 to about 2000 of 2-10 mparticles per ml, about 50 to about 350 of ≥10 μm particles per ml andabout 0 to about 50 of ≥25 μm particles per ml. In yet another aspect,an amount of particles in a dose of an anti-α4β7 antibody formulation isabout 500 to about 100,000, about 1000 to about 5000 or about 1500 toabout 3000 of 2-10 μm particles per ml.

The viscosity of an anti-α4β7 antibody formulation can be controlled forsubcutaneous or intramuscular administration. The viscosity can beaffected by protein concentration and pH. For example, as the proteinconcentration increases, the viscosity can increase. An increase in pHcan decrease the viscosity of the anti-α4β7 antibody formulation. Insome protein formulations, sodium chloride is added to reduce theviscosity of the formulation. Additional components that can affectviscosity of an anti-α4β7 antibody formulation are amino acids such ashistidine and arginine.

An anti-α4β7 antibody formulation can be isotonic (e.g., 250-350 mOsm)or hypertonic (e.g., greater than 350 mOsm, greater than 450 mOsm,greater than 550 mOsm or greater than 650 mOsm), e.g., for subcutaneousor intramuscular administration. In one aspect, the anti-α4β7 antibodyformulation is not hypotonic, e.g., less than 250 mOsm. In anotheraspect, the anti-α4β7 antibody formulation is about 350 to about 400mOsm, about 400 to about 450 mOsm or about 350 to about 450 mOsm.

Instability leading to denaturation can be assessed by differentialscanning calorimetry (DSC). Antibodies have two melting temperatures(Tm) in DSC, e.g., Tm1 and Tm2. Certain excipients can affect thestability of the native anti-α4β7 antibody. A finding of a highermelting temperature when comparing formulations by DSC can indicate amore stable anti-α4β7 antibody formulation with the higher Tm. Forexample, at pH5.7, the Tm of an anti-α4β7 antibody formulation is lower,and thus less stable than at pH 6.5. In one aspect, Tm1 of an anti-α4β7antibody formulation is >60° C. In another aspect, the Tm1 of ananti-α4β7 antibody formulation is about 65° C. to about 70° C. or about69° C. In one aspect, Tm2 of an anti-α4β7 antibody formulation is >80°C. In another aspect, the Tm2 of an anti-α4β7 antibody formulation isabout 82° C. to about 88° C. or about 86° C.

In one embodiment, an anti-α4β7 antibody formulation has a bindingaffinity or EC50 value of about 60% to about 140% of the referencestandard anti-α4β7 antibody. In one aspect, an anti-α4β7 antibody in aformulation described herein binds to α4β7, e.g., on a cell (WO98/06248or U.S. Pat. No. 7,147,851), at a value of about 80% to about 120% ofthe reference standard. In another embodiment, an anti-α4β7 antibodyformulation has the ability to inhibit at least 50%, or at least 60% ofthe binding of a cell expressing α4β7 integrin to MAdCAM (e.g.,MAdCAM-1), e.g., a MAdCAM-Ig chimera (see U.S. Patent ApplicationPublication No. 20070122404, also for reference standard examples).

As noted above, freezing of the formulation is specifically contemplatedherein. Hence, the formulation can be tested for stability upon freezingand thawing. Accordingly, the antibody in a liquid formulation may bestable upon freezing and thawing the formulation, for example theantibody can be stable after one, two, three, four, five or morefreeze/thaw cycles.

In some embodiments, the pharmaceutical formulation is a liquidformulation comprising at least about 60 mg/ml to about 170 mg/mlanti-α4β7 antibody, a buffering agent (e.g., histidine), and at leastabout 5 mM citrate. In other embodiments, the formulation is a liquidformulation comprising at least about 60 mg/ml to about 170 mg/mlanti-α4β7 antibody, a buffering agent (e.g., citrate), amino acid (e.g.,arginine) and surfactant (e.g., polysorbate 80).

In another embodiment, the formulation comprises at least about 140mg/ml or about 150 mg/ml to about 170 mg/ml, for example, about 160mg/ml of an anti-α4β7 antibody, a buffering agent (e.g., histidine), atleast about 5 mM citrate and a free amino acid (e.g., arginine).

In yet another embodiment, the formulation comprises at least about 160mg/ml of an anti-α4β7 antibody, a buffering agent (e.g., histidine), atleast about 5 mM citrate, 0.2% polysorbate 80, and a free amino acid(e.g., arginine). In an embodiment, the buffer concentration in theformulation is about 15 to about 75 mM, about 25 to about 65 mM, orabout 50 mM. The free amino acid concentration in the formulation isabout 50 to about 250 mM, about 75 to about 200 mM, about 100 to about150 mM or about 125 mM; the polysorbate 80 concentration in theformulation is about 0.05% to 0.4%, about 0.1% to 0.4%, about 0.1% to0.3%, about 0.1% to 0.25%, about 0.1% to 0.2%, or about 0.2%.

In some embodiments, the formulation is a solid formulation (e.g., alyophilized formulation), comprising a mixture of an anti-α4β7 antibody,citrate, histidine, arginine, polysorbate 80, and a lyoprotectant or asaccharide, such as a non-reducing sugar. Saccharide can be included inthe liquid formulation to reach concentrations from 0% to 20%, or about6% to about 10%.

In one embodiment, the formulation is lyophilized and stored as a singledose in one container, e.g., vial, syringe, cartridge, and/orautoinjector. The container can be stored at about 2-8° C. or 25° C.until it is administered to a subject in need thereof. The vial may forexample be a 5, 10 or 20 cc vial (for example for a 160 mg/ml dose). Thevial may contain at least about 20 mg, at least about 50 mg, at leastabout 70 mg, at least about 80 mg, at least about 100 mg, at least about120 mg, at least about 155 mg, at least about 180 mg, at least about 200mg, at least about 240 mg, at least about 300 mg, at least about 360 mg,at least about 400 mg, at least about 540 mg, or at least about 900 mgof anti-α4β7 antibody. In one aspect, the container contains about 165mg of anti-α4β7 antibody.

In another embodiment, the formulation is liquid and stored as a singledose in one or two vials, cartridges, syringes, or autoinjectors. Thevial, cartridge, syringe, or autoinjector can be stored at about 2-8° C.until its contents, e.g., an anti-α4β7 antibody, are administered to asubject in need thereof. The vial may, for example, be a 5, 10 or 20 ccvial (for example for a 160 mg/ml dose). The vial may contain at leastabout 20 mg, at least about 50 mg, at least about 70 mg, at least about80 mg, at least about 100 mg, at least about 120 mg, at least about 155mg, at least about 180 mg, at least about 200 mg, at least about 240 mg,at least about 300 mg, at least about 360 mg, at least about 400 mg, atleast about 540 mg, or at least about 900 mg of anti-α4β7 antibody. Inone aspect, the vial contains about 165 mg of anti-α4β7 antibody. Thesyringe or cartridge may be a 1 mL or 2 mL container (for example for a160 mg/mL dose) or more than 2 ml, e.g., for a higher dose (at least 320mg or 400 mg or higher). The syringe or cartridge may contain at leastabout 20 mg, at least about 50 mg, at least about 70 mg, at least about80 mg, at least about 100 mg, at least about 120 mg, at least about 155mg, at least about 180 mg, at least about 200 mg, at least about 240 mg,at least about 300 mg, at least about 360 mg, at least about 400 mg, orat least about 500 mg of anti-α4β7 antibody.

One or more other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington: The Science andPractice of Pharmacy, 21st Edition, Hendrickson, R. Ed. (2005) may beincluded in the formulation provided that they do not adversely affectthe desired characteristics of the formulation. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed and include; additional buffering agents;co-solvents; antioxidants including citrate and cysteine; chelatingagents such as EDTA; metal complexes (e.g., Zn-protein complexes);biodegradable polymers such as polyesters; preservatives; container walllubricants, e.g., silicone, mineral oil, glycerin, or TRIBOGLIDE@ (TriboFilm Research, Inc.) perfluoropolyether derivative, for injection easeand/or salt-forming counterions such as sodium.

α4β7 Antibodies

Anti-α4β7 antibodies suitable for use in the formulations includeantibodies from any desired source, such as fully human antibodies,murine antibodies, rabbit antibodies and the like, and any desiredengineered antibodies, such as chimeric antibodies, humanizedantibodies, and the like. Antigen-binding fragments of any of thesetypes of antibodies, such as Fab, Fv, scFv, Fab′ and F(ab′)₂ fragments,are also suitable for use in the formulations.

The anti-α4β7 antibody can bind to an epitope on the α4 chain (e.g.,humanized MAb 21.6 (Bendig et al., U.S. Pat. No. 5,840,299)), on the β7chain (e.g., FIB504 or a humanized derivative (e.g., Fong et al., U.S.Pat. No. 7,528,236)), or to a combinatorial epitope formed by theassociation of the α4 chain with the β7 chain. In one aspect, theantibody binds a combinatorial epitope on the α4β7 complex, but does notbind an epitope on the α4 chain or the β7 chain unless the chains are inassociation with each other. The association of α4 integrin with β7integrin can create a combinatorial epitope for example, by bringinginto proximity residues present on both chains which together comprisethe epitope or by conformationally exposing on one chain, e.g., the α4integrin chain or the β7 integrin chain, an epitopic binding site thatis inaccessible to antibody binding in the absence of the properintegrin partner or in the absence of integrin activation. In anotheraspect, the anti-α4β7 antibody binds both the α4 integrin chain and theβ7 integrin chain, and thus, is specific for the α4β7 integrin complex.Such antibodies can bind α4β7 but not bind α4β1, and/or not bindα_(E)β7, for example. In another aspect, the anti-α4β7 antibody binds tothe same or substantially the same epitope as the Act-1 antibody(Lazarovits, A. I. et al., J. Immunol., 133(4): 1857-1862 (1984),Schweighoffer et al., J. Immunol., 151(2): 717-729, 1993; Bednarczyk etal., J. Biol. Chem., 269(11): 8348-8354, 1994). Murine ACT-1 Hybridomacell line, which produces the murine Act-1 monoclonal antibody, wasdeposited under the provisions of the Budapest Treaty on Aug. 22, 2001,on behalf of Millennium Pharmaceuticals, Inc., 40 Landsdowne Street,Cambridge, Mass. 02139, U.S.A., at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., underAccession No. PTA-3663. In another aspect, the anti-α4β7 antibody is ahuman antibody or an α4β7 binding protein using the CDRs provided inU.S. Patent Application Publication No. 2010/0254975.

In one aspect, the anti-α4β7 antibody inhibits binding of α4β7 to one ormore of its ligands (e.g. the mucosal addressin, e.g., MAdCAM (e.g.,MAdCAM-1), fibronectin, and/or vascular addressin (VCAM)). PrimateMAdCAMs (e.g., MAdCAM-1) are described in the PCT publication WO96/24673, the entire teachings of which are incorporated herein by thisreference. In another aspect, the anti-α4β7 antibody inhibits binding ofα4β7 to MAdCAM (e.g., MAdCAM-1) and/or fibronectin without inhibitingthe binding of VCAM.

In one aspect, the anti-α4β7 antibodies for use in the formulations arehumanized versions of the mouse Act-1 antibody. Suitable methods forpreparing humanized antibodies are well-known in the art. Generally, thehumanized anti-α4β7 antibody will contain a heavy chain that containsthe 3 heavy chain complementarity determining regions (CDRs, CDR1, SEQID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) of the mouse Act-1antibody and suitable human heavy chain framework regions; and alsocontain a light chain that contains the 3 light chain CDRs (CDR1, SEQ IDNO:11, CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13) of the mouse Act-1antibody and suitable human light chain framework regions. The humanizedAct-1 antibody can contain any suitable human framework regions,including consensus framework regions, with or without amino acidsubstitutions. For example, one or more of the framework amino acids canbe replaced with another amino acid, such as the amino acid at thecorresponding position in the mouse Act-1 antibody. The human constantregion or portion thereof, if present, can be derived from the κ or λlight chains, and/or the γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g., α1, α2),δ or ε heavy chains of human antibodies, including allelic variants. Aparticular constant region (e.g., IgG1), variant or portions thereof canbe selected in order to tailor effector function. For example, a mutatedconstant region (variant) can be incorporated into a fusion protein tominimize binding to Fc receptors and/or ability to fix complement (seee.g., Winter et al., GB 2,209,757 B; Morrison et al., WO 89/07142;Morgan et al., WO 94/29351, Dec. 22, 1994). Humanized versions of Act-1antibody were described in PCT publications nos. WO98/06248 andWO07/61679, the entire teachings of each of which are incorporatedherein by this reference.

In another aspect, the anti-α4β7 humanized antibodies for use in theformulation comprise a heavy chain variable region comprising aminoacids 20 to 140 of SEQ ID NO:2, and a light chain variable regioncomprising amino acids 20 to 131 of SEQ ID NO:4 or amino acids 21 to 132of SEQ ID NO:5. If desired, a suitable human constant region(s) can bepresent. For example, the humanized anti-α4β7 antibody can comprise aheavy chain that comprises amino acids 20 to 470 of SEQ ID NO:2 and alight chain comprising amino acids 21 to 239 of SEQ ID NO:5. In anotherexample, the humanized anti-α4β7 antibody can comprise a heavy chainthat comprises amino acids 20 to 470 of SEQ ID NO:2 and a light chaincomprising amino acids 20 to 238 of SEQ ID NO:4. FIG. 4 shows analignment which compares the generic light chains of human antibodieswith murine antibodies. The alignment illustrates that the humanizedlight chain of vedolizumab (e.g., Chemical Abstract Service (CAS,American Chemical Society) Registry number 943609-66-3), with two mouseresidues switched for human residues, is more human than the light chainof LDP-02 (FIG. 3). In addition, LDP-02 has the somewhat hydrophobic,flexible alanine 114 and a hydrophilic site (Aspartate 115) that isreplaced in vedolizumab with the slightly hydrophilichydroxyl-containing threonine 114 and hydrophobic, potentially inwardfacing valine 115 residue.

Further substitutions to the antibody sequence can be, for example,mutations to the heavy and light chain framework regions, such as amutation of isoleucine to valine on residue 2 of SEQ ID NO:14; amutation of methionine to valine on residue 4 of SEQ ID NO:14; amutation of alanine to glycine on residue 24 of SEQ ID NO:15; a mutationof arginine to lysine at residue 38 of SEQ ID NO:15; a mutation ofalanine to arginine at residue 40 of SEQ ID NO:15; a mutation ofmethionine to isoleucine on residue 48 of SEQ ID NO:15; a mutation ofisoleucine to leucine on residue 69 of SEQ ID NO:15; a mutation ofarginine to valine on residue 71 of SEQ ID NO:15; a mutation ofthreonine to isoleucine on residue 73 of SEQ ID NO:15; or anycombination thereof; and replacement of the heavy chain CDRs with theCDRs (CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) ofthe mouse Act-1 antibody; and replacement of the light chain CDRs withthe light chain CDRs (CDR1, SEQ ID NO:11, CDR2, SEQ ID NO:12 and CDR3,SEQ ID NO:13) of the mouse Act-1 antibody.

In some embodiments, the anti-α4β7 humanized antibodies for use in theformulation comprise a heavy chain variable region that has about 95%,96%, 97%, 98%, or 99% sequence identity to amino acids 20 to 140 of SEQID NO:2, and a light chain variable region that has about 95%, 96%, 97%,98%, or 99% sequence identity to amino acids 20 to 131 of SEQ ID NO:4 oramino acids 21 to 132 of SEQ ID NO:5. Amino acid sequence identity canbe determined using a suitable sequence alignment algorithm, such as theLasergene system (DNASTAR, Inc., Madison, Wis.), using the defaultparameters. In an embodiment, the anti-α4β7 antibody for use in theformulation is vedolizumab (CAS, American Chemical Society, Registrynumber 943609-66-3).

Other α4β7 antibodies may also be used in the formulations and dosingregimes described herein. For example, the α4β7 antibodies described inUS 2010/0254975 (Amgen, Inc.), incorporated by reference herein in itsentirety, are suitable for use in the formulations and methods oftreating inflammatory bowel disease in an individual.

The anti-α4β7 antibody can be produced by expression of nucleic acidsequences encoding each chain in living cells, e.g., cells in culture. Avariety of host-expression vector systems may be utilized to express theantibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an anti-α4β7 antibody in situ. These include but arenot limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3, NS0 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Forexample, mammalian cells such as Chinese hamster ovary cells (CHO), inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus, is an effective expressionsystem for antibodies (Foecking et al., Gene 45:101 (1986); Cockett etal., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to Chinese hamster ovary (CHO), NS0,HeLa, VERY, baby hamster kidney (BHK), monkey kidney (COS), MDCK, 293,3T3, WI38, human hepatocellular carcinoma cells (e.g., Hep G2), breastcancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 andT47D, and normal mammary gland cell line such as, for example, CRL7030and Hs578Bst.

The glycosylation machinery of different cell types can produceantibodies with different glycosylation composition than in another celltype, or no glycosylation, as with bacterial cells. In one aspect, celltypes for production of the anti-α4β7 antibody are mammalian cells, suchas NS0 or CHO cells. In one aspect, the mammalian cells can comprise thedeletion of an enzyme involved in cell metabolism and the exogenous geneof interest can be operably linked to a replacement enzyme, e.g., in aconstruct or vector for introduction into the cells, e.g., bytransformation or transfection. The construct or vector with theexogenous gene confers to the cells which host the construct or vector aselection advantage to encourage production of the polypeptide encodedby the exogenous gene. In one embodiment, CHO cells are DG44 cells(Chasin and Urlaub (1980) PNAS USA 77:4216), comprising the deletion orinactivation of the dihydrofolate reductase gene. In another embodiment,CHO cells are CHO K1 cells comprising the deletion or inactivation ofthe glutamine synthase gene (see, e.g., U.S. Pat. No. 5,122,464 or5,827,739).

Solid Formulations

Solid formulations of the invention are generally prepared by drying aliquid formulation. Any suitable method of drying can be used, such aslyophilization or spray drying. In one aspect, a lyoprotectant is addedto the formulation prior to lyophilization. Lyophilization involvesfreezing a liquid formulation, usually in the container that will beused to store, ship and distribute the formulation (e.g., a vial,syringe (e.g., a single- or dual-chamber syringe), or cartridge (e.g., asingle- or dual-chamber cartridge) (See, e.g., Gatlin and Nail inProtein Purification Process Engineering, ed. Roger G. Harrison, MarcelDekker Inc., 317-367 (1994).) Once the formulation is frozen, theatmospheric pressure is reduced and the temperature is adjusted to allowremoval of the frozen solvent e.g., through sublimation. This step ofthe lyophilization process is sometimes referred to as primary drying.If desired, the temperature can then be raised to remove any solventthat is still bound to the dry formulation by evaporation. This step ofthe lyophilization process is sometimes referred to as secondary drying.When the formulation has reached the desired degree of dryness, thedrying process is concluded and the containers are sealed. The finalsolid formulation is sometimes referred to as a “lyophilizedformulation” or a “cake.” The lyophilization process can be performedusing any suitable equipment. Suitable lyophilization equipment isavailable from a number of commercial sources (e.g., SP Scientific,Stone Ridge, N.Y.).

A variety of suitable apparatuses can be used to dry liquid formulationsto produce a solid (e.g., lyophilized) formulation. Generally,lyophilized formulations are prepared by those of skill in the art usinga sealed chamber that contains shelves, on which vials of the liquidformulation to be dried are placed. The temperature of the shelves, aswell as cooling and heating rate can be controlled, as can the pressureinside the chamber. It will be understood that various processparameters discussed herein refer to processes performed using this typeof apparatus. Persons of ordinary skill can easily adapt the parametersdescribed herein to other types of drying apparatuses if desired.

Suitable temperatures and the amount of vacuum for primary and secondarydrying can be readily determined by a person of ordinary skill. Ingeneral, the formulation is frozen at a temperature of about −30° C. orless, such as −40° C. or −50° C. The rate of cooling can affect theamount and size of ice crystals in the matrix. Primary drying isgenerally conducted at a temperature that is about 10° C., about 20° C.,about 30° C., about 40° C. or about 50° C. warmer than the freezingtemperature. In one aspect, the primary drying conditions can be set tomaintain the anti-α4β7 antibody below the glass transition temperatureor collapse temperature of the formulation. Above the collapsetemperature, the amorphous frozen matrix can flow (collapse), with aresult that the protein molecules may not be surrounded by a rigid,solid matrix, and the protein molecules may not be stable in thecollapsed matrix. Also, the formulation can be difficult to fully dry ifcollapse occurs. The resulting higher amounts of moisture in theformulation can lead to higher rates of protein degradation and adecrease in the amount of time that the lyophilized product can bestored before its quality diminishes to unacceptable levels. In oneaspect, the shelf temperature and chamber pressure are selected tomaintain the product temperature below the collapse temperature duringprimary drying. The glass transition temperature of a frozen formulationcan be measured by methods known in the art, e.g., by differentialscanning calorimetry (DSC). The collapse temperature can be measured bymethods known in the art, e.g. freeze-drying microscopy, opticalcoherence tomography. The drying step can remove at least 50%, at least60%, at least 70% or more of the solvent. In one aspect, the primarydrying step removes more than 80% of the solvent from the anti-α4β7antibody formulation.

Vial size can be selected based on the surface area which will beexposed to the shelf and to the vacuum during lyophilization. Dryingtime is directly proportional to cake height, thus the vial size may bechosen based upon what is determined to be a reasonable cake height. Avial with a large diameter relative to volume can provide a high amountof contact with the shelf for efficient heat transfer during thelyophilization cycle. A dilute antibody solution in a high volume ofliquid will require more time for drying. A balance in vial size versusformulation volume needs to be struck, because larger vials can be moreexpensive to store and ship and have a larger headspace to formulationratio, and may expose a high proportion of the formulation to thedegradative effects of moisture during long term storage. For a 165 mgdose, the vial size of the anti-α4β7 antibody formulation can be 3 mL, 5ml or 10 ml. In one aspect, the vial size is 5 ml for a 160 mg/mlsolution.

The principles for choosing a cartridge or syringe size forlyophilization are similar to that of the vial. The depth of the cakeheight will also increase the drying time as the height increases. Thediameter and size of the syringe or cartridge must be balanced out withthe final formulation volume. Larger diameters can increase the rate ofmoisture uptake in the lyophilized cake, thus increasing the degradativeeffects of moisture during storage. For a 165 mg dose, the anti-α4β7antibody formulation volume can be 1 ml or 2 mL. In one aspect, thesyringe or cartridge size is greater than 1 mL for a 160 mg/mL solution.

After lyophilization, the vial, syringe, or cartridge can be sealed,e.g., stoppered, under a vacuum. Alternatively, a gas, e.g., dry air ornitrogen, can be allowed into the container prior to sealing. Whereoxidation is a concern, the gas allowed into the lyophilization chambercan comprise a gas which retards or prevents oxidation of thelyophilized product. In one aspect the gases are non-oxygenated gases,e.g., nitrogen, or an inert gas, e.g., helium, neon, argon, krypton orxenon. In another aspect, the gas is nitrogen or argon.

Treatment with the Antibody Formulation

In one aspect, the invention provides a method of treating a disease ordisorder in a subject comprising administering to a subject theanti-α4β7 antibody formulation described herein in an amount effectiveto treat the disease or disorder, e.g., in humans. The human subject maybe an adult (e.g., 18 years or older), an adolescent, or a child. Thehuman subject may be a person 65 years or older. In contrast toalternative therapeutic dosing regimens, a human subject 65 years orolder does not require any modification of the dosing regimen describedherein, and may be administered the conventional anti-α4β7 antibodyformulation described herein.

The subject may have had a lack of an adequate response with, loss ofresponse to, or was intolerant to treatment with an immunomodulator, aTNF-α antagonist, or combinations thereof. The patient may havepreviously received treatment with at least one corticosteroid (e.g.,prednisone) for the inflammatory bowel disease. An inadequate responseto corticosteroids refers to signs and symptoms of persistently activedisease despite a history of at least one 4-week induction regimen thatincluded a dose equivalent to prednisone 30 mg daily orally for 2 weeksor intravenously for 1 week. A loss of response to corticosteroidsrefers to two failed attempts to taper corticosteroids to below a doseequivalent to prednisone 10 mg daily orally. Intolerance ofcorticosteroids includes a history of Cushing's syndrome,osteopenia/osteoporosis, hyperglycemia, insomnia and/or infection.

An immunomodulator may be, for example, oral azathioprine,6-mercaptopurine, or methotrexate. An inadequate response to animmunomodulator refers to signs and symptoms of persistently activedisease despite a history of at least one 8 week regimen or oralazathioprine (≥1.5 mg/kg), 6-mercaptopurine (≥0.75 mg/kg), ormethotrexate (≥12.5 mg/week). Intolerance of an immunomodulatorincludes, but is not limited to, nausea/vomiting, abdominal pain,pancreatitis, LFT abnormalities, lymphopenia, TPMT genetic mutationand/or infection.

A TNF-α antagonist is, for example, an agent that inhibits thebiological acitivity of TNF-α, and preferably binds TNF-α, such as amonoclonal antibody, e.g., REMICADE (infliximab), HUMIRA (adalimumab),CIMZIA (certolizumab pegol), SIMPONI (golimumab) or a circulatingreceptor fusion protein such as ENBREL (etanercept). An inadequateresponse to a TNF-α antagonist refers to signs and symptoms ofpersistently active disease despite a history of at least one 4 weekinduction regimen of infliximab 5 mg/kg IV, 2 doses at least 2 weeksapart; one 80 mg subcutaneous dose of adalimumab, follwed by one 40 mgdose at least two weeks apart; or 400 mg subcutaneously of certolizumabpegol, 2 doses at least 2 weeks apart. A loss of response to a TNF-αantagonist refers to recurrence of symptoms during maintenance dosingfollowing prior clinical benefit. Intolerance of a TNF-α antagonistincludes, but is not limited to infusion related reaction,demyelination, congestive heart failure, and/or infection.

A loss of maintenance of remission, as used herein for ulcerativecolitis subjects, refers to an increase in Mayo score of at least 3points and a Modified Baron Score of at least 2.

In another aspect, the present invention provides anti-α4β7 antibodyformulations which (1) can bind α4β7 integrin in vitro and/or in vivo;and (2) can modulate an activity or function of an α4β7 integrin, suchas (a) binding function (e.g., the ability of α4β7 integrin to bind toMAdCAM (e.g., MAdCAM-1), fibronectin and/or VCAM-1) and/or (b) leukocyteinfiltration function, including recruitment and/or accumulation ofleukocytes in tissues (e.g., the ability to inhibit lymphocyte migrationto intestinal mucosal tissue). In one embodiment, an antibody in theformulation can bind an α4β7 integrin, and can inhibit binding of theα4β7 integrin to one or more of its ligands (e.g., MAdCAM (e.g.,MAdCAM-1), VCAM-1, fibronectin), thereby inhibiting leukocyteinfiltration of tissues (including recruitment and/or accumulation ofleukocytes in tissues). In another embodiment, an antibody in theformulation can bind an α4β7 integrin, and can selectively inhibitbinding of the α4β7 integrin to one or more of its ligands (e.g., MAdCAM(e.g., MAdCAM-1), VCAM-1, fibronectin), thereby inhibiting leukocyteinfiltration of tissues (including recruitment and/or accumulation ofleukocytes in tissues). Such anti-α4β7 antibody formulations can inhibitcellular adhesion of cells bearing an α4β7 integrin to vascularendothelial cells in mucosal tissues, including gut-associated tissues,lymphoid organs or leukocytes (especially lymphocytes such as T or Bcells) in vitro and/or in vivo. In yet another embodiment, the anti-α4β7antibody formulation of the present invention can inhibit theinteraction of α4β7 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin. Instill yet another embodiment, the anti-α4β7 antibody formulation of thepresent invention can inhibit the interaction of α4β7 with MAdCAM (e.g.,MAdCAM-1) and/or fibronectin selectively, e.g., without inhibiting theinteraction of α4β7 with VCAM.

The anti-α4β7 antibody formulations of the present invention can be usedto modulate (e.g., inhibit (reduce or prevent)) binding function and/orleukocyte (e.g., lymphocyte, monocyte) infiltration function of α4β7integrin. For example, humanized immunoglobulins which inhibit thebinding of α4β7 integrin to a ligand (i.e., one or more ligands) can beadministered according to the method in the treatment of diseasesassociated with leukocyte (e.g., lymphocyte, monocyte) infiltration oftissues (including recruitment and/or accumulation of leukocytes intissues), particularly of tissues which express the molecule MAdCAM(e.g., MAdCAM-1).

An effective amount of an anti-α4β7 antibody formulation of the presentinvention (i.e., one or more) is administered to an individual (e.g., amammal, such as a human or other primate) in order to treat such adisease. For example, inflammatory diseases, including diseases whichare associated with leukocyte infiltration of the gastrointestinal tract(including gut-associated endothelium), other mucosal tissues, ortissues expressing the molecule MAdCAM (e.g., MAdCAM-1) (e.g.,gut-associated tissues, such as venules of the lamina propria of thesmall and large intestine; and mammary gland (e.g., lactating mammarygland)), can be treated according to the present method. Similarly, anindividual having a disease associated with leukocyte infiltration oftissues as a result of binding of leukocytes to cells (e.g., endothelialcells) expressing MAdCAM (e.g., MAdCAM-1) can be treated according tothe present invention.

In one embodiment, diseases which can be treated accordingly includeinflammatory bowel disease (IBD), such as ulcerative colitis, Crohn'sdisease, ileitis, Celiac disease, nontropical Sprue, enteropathyassociated with seronegative arthropathies, microscopic or collagenouscolitis, eosinophilic gastroenteritis, or pouchitis resulting afterproctocolectomy, and ileoanal anastomosis. In some embodiments, theinflammatory bowel disease is Crohn's disease or ulcerative colitis. Theulcerative colitis may be moderate to severely active ulcerativecolitis. Treatment may result in mucosal healing in patients sufferingfrom moderate to severely active ulcerative colitis. Treatment may alsoresult in a reduction, elimination, or reduction and elimination ofcorticosteroid use by the patient.

Pancreatitis and insulin-dependent diabetes mellitus are other diseaseswhich can be treated using the formulations of the invention. It hasbeen reported that MAdCAM (e.g., MAdCAM-1) is expressed by some vesselsin the exocrine pancreas from NOD (nonobese diabetic) mice, as well asfrom BALB/c and SJL mice. Expression of MAdCAM (e.g., MAdCAM-1) wasreportedly induced on endothelium in inflamed islets of the pancreas ofthe NOD mouse, and MAdCAM (e.g., MAdCAM-1) was the predominant addressinexpressed by NOD islet endothelium at early stages of insulitis(Hanninen, A., et al., J. Clin. Invest., 92: 2509-2515 (1993)).Treatment of NOD mice with either anti-MAdCAM or anti-β7 antibodiesprevented the development of diabetes (Yang et al., Diabetes,46:1542-1547 (1997)). Further, accumulation of lymphocytes expressingα4β7 within islets was observed, and MAdCAM-1 was implicated in thebinding of lymphoma cells via α4β7 to vessels from inflamed islets(Hanninen, A., et al., J. Clin. Invest., 92: 2509-2515 (1993)) or to thegastrointestinal tract in mantle cell lymphoma (Geissmann et al., Am. J.Pathol., 153:1701-1705 (1998)).

Examples of inflammatory diseases associated with mucosal tissues whichcan be treated using a formulation of the invention includecholecystitis, cholangitis (Adams and Eksteen Nature Reviews 6:244-251(2006) Grant et al., Hepatology 33:1065-1072 (2001)), e.g., primarysclerosing cholangitis, Behcet's disease, e.g., of the intestine, orpericholangitis (bile duct and surrounding tissue of the liver), andgraft versus host disease (e.g., in the gastrointestinal tract (e.g.,after a bone marrow transplant) (Petrovic et al. Blood 103:1542-1547(2004)). As seen in Crohn's disease, inflammation often extends beyondthe mucosal surface, accordingly chronic inflammatory diseases, such assarcoidosis, chronic gastritis, e.g., autoimmune gastritis (Katakai etal., Int. Immunol., 14:167-175 (2002)) and other idiopathic conditionscan be amenable to treatment.

The invention also relates to a method of inhibiting leukocyteinfiltration of mucosal tissue. The invention also relates to a methodfor treating cancer (e.g., an α4β7 positive tumor, such as a lymphoma).Other examples of inflammatory diseases associated with mucosal tissueswhich can be treated using a formulation of the invention includemastitis (mammary gland) and irritable bowel syndrome.

Diseases or pathogens whose etiologies exploit the interaction of MAdCAM(e.g., MAdCAM-1) with α4β7 can be treated with an anti-α4β7 antibody ina formulation described herein. Examples of such diseases includeimmunodeficiency disorders, such as caused by human immunodeficiencyvirus (See, e.g., WO2008140602).

A formulation of the invention is administered in an effective amountwhich inhibits binding of α4β7 integrin to a ligand thereof. Fortherapy, an effective amount will be sufficient to achieve the desiredtherapeutic (including prophylactic) effect (such as an amountsufficient to reduce or prevent α4β7 integrin-mediated binding and/orsignaling, thereby inhibiting leukocyte adhesion and infiltration and/orassociated cellular responses). An effective amount of an anti-α4β7antibody, e.g., an effective titer sufficient to maintain saturation,e.g., neutralization, of α4β7 integrin, can induce clinical response orremission in inflammatory bowel disease. An effective amount of ananti-α4β7 antibody can lead to mucosal healing in ulcerative colitis orCrohn's disease. A formulation of the invention can be administered in aunit dose or multiple doses. The dosage can be determined by methodsknown in the art and can be dependent, for example, upon theindividual's age, sensitivity, tolerance and overall well-being.Examples of modes of administration include topical routes such as nasalor inhalational or transdermal administration, enteral routes, such asthrough a feeding tube or suppository, and parenteral routes, such asintravenous, intramuscular, subcutaneous, intraarterial,intraperitoneal, or intravitreal administration. Suitable dosages forantibodies can be from about 0.1 mg/kg body weight to about 10.0 mg/kgbody weight per treatment, for example about 2 mg/kg to about 7 mg/kg,about 3 mg/kg to about 6 mg/kg, or about 3.5 to about 5 mg/kg. Inparticular embodiments, the dose administered is about 0.3 mg/kg, about0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg,about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9mg/kg, or about 10 mg/kg. The total dose may be about 22 mg, about 50mg, about 72 mg, about 125 mg, about 165 mg, or about 432 mg. The totaldose may be at least 77 mg, at least 125 mg or at least 356 mg. In oneembodiment, the total dose is 165 mg. In another embodiment, the totaldose is 108 mg. In another embodiment, the total dose is 216 mg.

Modeling and simulations (BERKELEY MADONNA™ software, University ofCalifornia) using pharmacokinetic (PK) data from studies of availabilityof anti-α4β7 antibody over time after administration can assesspotential dosing regimens for subcutaneous or intramuscularadministration. PK data can be evaluated for induction and formaintenance regimens. Another modeling approach is populationpharmacokinetic/pharmacodynamic analysis (NONMEM® nonlinear mixedeffects modeling tool, ICON plc, Dublin, Ireland). Both exposure levelsand trough levels can be analyzed.

Typically, after target, e.g., α4β7 integrin, saturation is reached, theantibody concentration in the blood has a linear relationship to thedose administered. An anti-α4β7 antibody administered by thesubcutaneous or intramuscular route has about 60% to about 90% of thebioavailability of an anti-α4β7 antibody administered by an intravenousroute. In an example of this relationship, if an IV dose is assumed tohave a 100% bioavalability and a subcutaneous dose is found to have a69.5% bioavailability, then a 300 mg intravenous dose can be matchedwith a 432 mg dose by subcutaneous administration. Accordingly, a 150 mgintravenous dose can be matched by a 216 mg subcutaneous dose at 69.5%relative bioavailability. Similarly, if a subcutaneous dose is found tohave a 75% availability and an intramuscular dose is found to have an80% bioavailability, then to match a 300 mg intravenous dose, thesubcutaneous dose can be 400 mg and the intramuscular dose can be 375mg. Tables 40-43 in the examples illustrate these relationships andprovide useful doses and dosing regimens of an anti-α4β7 antibody.

In some aspects, the dosing regimen has two phases, an induction phaseand a maintenance phase. In the induction phase, the antibody orantigen-binding fragment thereof is administered in a way that quicklyprovides an effective amount of the antibody or antigen binding fragmentthereof suitable for certain purposes, such as inducing immune toleranceto the antibody or antigen-binding fragment thereof or for inducing aclinical response and ameliorating inflammatory bowel disease symptoms.A patient can be administered an induction phase treatment when firstbeing treated by an anti-α4β7 antibody, when being treated after a longabsence from therapy, e.g., more than three months, more than fourmonths, more than six months, more than nine months, more than one year,more than eighteen months or more than two years since anti-α4β7antibody therapy or during maintenance phase of anti-α4β7 antibodytherapy if there has been a return of inflammatory bowel diseasesymptoms, e.g., a relapse from remission of disease. In someembodiments, the induction phase regimen results in a higher mean troughserum concentration, e.g., the concentration just before the next dose,than the mean steady state trough serum concentration maintained duringthe maintenance regimen.

In the maintenance phase, the antibody or antigen-binding fragmentthereof is administered in a way that continues the response achieved byinduction therapy with a stable level of antibody or antigen-bindingfragment thereof. A maintenance regimen can prevent return of symptomsor relapse of inflammatory bowel disease. A maintenance regimen canprovide convenience to the patient, e.g., be a simple dosing regimen orrequire infrequent trips for treatment. In some embodiments, themaintenance regimen can include administration of the anti-α4β7 antibodyor antigen-binding fragment thereof, e.g., in a formulation describedherein, by a strategy selected from the group consisting of low dose,infrequent administration, self-administration and a combination any ofthe foregoing.

In one embodiment, e.g., during an induction phase of therapy, thedosing regimen provides an effective amount of an anti-α4β7 antibody orantigen-binding fragment in a formulation described herein for inducingremission of an inflammatory bowel disease in a human patient. In someembodiments, the effective amount of the anti-α4β7 antibody issufficient to achieve about 5 g/ml to about 60 g/ml, about 15 μg/ml toabout 45 μg/ml, about 20 μg/ml to about 30 μg/ml, or about 25 μg/ml toabout 35 μg/ml mean trough serum concentration of the anti-α4β7 antibodyby the end of the induction phase. The duration of induction phase canbe about four weeks, about five weeks, about six weeks, about sevenweeks, or about eight weeks of treatment. In some embodiments, theinduction regimen can utilize a strategy selected from the groupconsisting of high dose, frequent administration, and a combination ofhigh dose and frequent administration of the anti-α4β7 antibody orantigen-binding fragment thereof, e.g., in a formulation describedherein. Induction dosing can be once, or a plurality of more than onedose, e.g., at least two doses. During induction phase, a dose can beadministered once per day, every other day, twice per week, once perweek, once every ten days, once every two weeks or once every threeweeks. In some embodiments, the induction doses are administered withinthe first two weeks of therapy with the anti-α4β7 antibody. In oneembodiment, induction dosing can be once at initiation of treatment (day0) and once at about two weeks after initiation of treatment. In anotherembodiment, the induction phase duration is six weeks. In anotherembodiment, the induction phase duration is six weeks and a plurality ofinduction doses are administered during the first two weeks.

In some embodiments, e.g., when initiating treatment of a patient withsevere inflammatory bowel disease (e.g., in patients who have failedanti-TNFα therapy), the induction phase needs to have a longer durationthan for patients with mild or moderate disease. In some embodiments,the induction phase for a patient with a severe disease can have aduration of at least 6 weeks, at least 8 weeks, at least 10 weeks, atleast 12 weeks or at least 14 weeks. In one embodiment, an inductiondosing regimen for a patient with a severe disease can include a dose atweek 0 (initiation of treatment), a dose at week 2 and a dose at week 6.In another embodiment, an induction dosing regimen for a patient with asevere disease can comprise a dose at week 0 (initiation of treatment),a dose at week 2, a dose at week 6 and a dose at week 10.

In one embodiment, e.g., during a maintenance phase of therapy, thedosing regimen maintains a mean steady state trough serum concentration,e.g., the plateau concentration just before the next dose, of about 5 toabout 25 μg/mL, about 7 to about 20 μg/mL, about 5 to about 10 μg/mL,about 10 to about 20 μg/mL, about 15 to about 25 μg/mL or about 9 toabout 13 μg/mL of anti-α4β7 antibody. In another embodiment, the dosingregimen e.g., during a maintenance phase of therapy, maintains a meansteady state trough serum concentration of about 20 to about 30 μg/mL,about 20 to about 55 μg/mL, about 30 to about 45 μg/mL, about 45 toabout 55 μg/mL or about 35 to about 40 μg/mL of anti-α4β7 antibody. Inanother embodiment, the dosing regimen e.g., during a maintenance phaseof therapy, maintains a long term mean serum concentration, e.g.,exposure (e.g., area under the curve-concentration-time) of about 15 toabout 40 μg/mL, about 10 to about 50 μg/mL, about 18 to about 26 μg/mL,or about 22 to about 33 μg/mL of anti-α4β7 antibody. In yet anotherembodiment, the dosing regimen e.g., during a maintenance therapy,maintains a long term mean serum concentration, e.g., exposure (e.g.,area under the curve-concentration-time) of about 35 to about 90 μg/mL,about 45 to about 75 μg/mL, about 52 to about 60 μg/mL or about 50 toabout 65 μg/mL of anti-α4β7 antibody.

The final dosage form can comprise the entire dose in about 0.5 ml, inabout 1 ml, in about 1.5 ml in about 2 ml, in about 2.5 ml, in about 3ml of the antibody formulation.

The final dosage form for intravenous administration may be at aconcentration of between about 1.0 mg/ml to about 1.4 mg/ml, about 1.0mg/ml to about 1.3 mg/ml, about 1.0 mg/ml to about 1.2 mg/ml, about 1.0to about 1.1 mg/ml, about 1.1 mg/ml to about 1.4 mg/ml, about 1.1 mg/mlto about 1.3 mg/ml, about 1.1 mg/ml to about 1.2 mg/ml, about 1.2 mg/mlto about 1.4 mg/ml, about 1.2 mg/ml to about 1.3 mg/ml, or about 1.3mg/ml to about 1.4 mg/ml. The final dosage form may be at aconcentration of about 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.1 mg/ml, about1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6mg/ml, about 1.8 mg/ml or about 2.0 mg/ml.

The dose can be administered once per week, once every 2 weeks, onceevery 3 weeks, once every 4 weeks, once every 6 weeks, once every 8weeks or once every 10 weeks. A higher or more frequent dose, e.g.,every other day, once per week, once every 2 weeks, once every 3 weeksor once every 4 weeks can be useful for inducing remission of activedisease or for treating a new patient, e.g., for inducing tolerance tothe anti-α4β7 antibody. A dose once every 2 weeks, once every 3 weeks,once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8weeks or once every 10 weeks, can be useful for preventative therapy,e.g., to maintain remission of a patient with chronic disease. In oneaspect, the treatment regimen is treatment at day 0, about week 2, aboutweek 6 and every 1 or 2 weeks thereafter. In another aspect, theinduction treatment regimen is treatment every other day for a total of6 treatments.

The dosing regimen can be optimized to induce a clinical response andclinical remission in the inflammatory bowel disease of the patient. Insome embodiments, the dosing regimen does not alter the ratio of CD4 toCD8 in cerebrospinal fluid of patients receiving treatment.

In some aspects, a durable clinical remission, for example, a clinicalremission which is sustained through at least two, at least three, atleast four visits with a caretaking physician within a six month or oneyear period after beginning treatment, may be achieved with an optimizeddosing regimen.

In some aspects, a durable clinical response, for example, a clinicalresponse which is sustained for at least 6 months, at least 9 months, atleast a year, after the start of treatment, may be achieved with anoptimized dosing regimen.

The formulation may be administered subcutaneously in single or multipleinjections. For example, the volume of a single injection may range fromabout 0.5 ml to about 3 ml. In an embodiment, the volume of a singleinjection may be about 0.6 ml to about 1.1 ml or about 1 ml to about 3ml. In one aspect, the volume of a single injection is about 1 ml. Thegauge of the needle used to administer the formulation subcutaneouslymay be about 25, about 26, about 27, about 28, about 29 or about 30 G.

The formulation may be administered intramuscularly in single ormultiple injections. For example, the volume of a single injection mayrange from about 0.5 ml to about 5 ml. In an embodiment, the volume of asingle injection may be about 2 ml to about 5 ml, about 0.6 ml to about1.1 ml or about 1 ml to about 3 ml. In one aspect, the volume of asingle injection is about 1 ml, about 2 ml, about 3 ml, about 4 ml, orabout 5 ml. The needle used to administer the formulationintramuscularly may be about ⅝″, about ⅞″, about 1″, about 1.25″, about1.5″, about 2″, or about 3″. The gauge of the needle may be between20-22 G for intramuscular administration.

In one aspect, the invention relates to a method for treating a humanpatient suffering from inflammatory bowel disease, wherein the methodcomprises the step of administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment thereof is administered to the patient according to thefollowing dosing regimen: (a) initial doses of 165 mg of the humanizedimmunoglobulin or antigen-binding fragment thereof as a subcutaneousinjection every other day for six doses; (b) followed by a seventh andsubsequent doses of 165 mg of the humanized immunoglobulin orantigen-binding fragment thereof as a subcutaneous injection every twoweeks or every four weeks as needed; wherein the dosing regimen inducesa clinical response and clinical remission in the inflammatory boweldisease of the patient; and further wherein the humanized immunoglobulinor antigen-binding fragment has binding specificity for the α4β7complex, wherein the antigen-binding region comprises threecomplementarity determining regions (CDR1, CDR2, and CDR3) of a lightchain variable region and three complementarity determining regions(CDR1, CDR2, and CDR3) of a heavy chain variable region of the aminoacid sequence set forth below: light chain: CDR1 SEQ ID NO: 11, CDR2 SEQID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQID NO: 9, CDR3 SEQ ID NO: 10.

In one aspect, the invention relates to a method for treating a humanpatient suffering from inflammatory bowel disease, wherein the methodcomprises the step of administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to the following dosing regimen: (a) an initialintravenous dose of 300 mg of the humanized immunoglobulin orantigen-binding fragment thereof as an intravenous infusion; (b)followed by a second intravenous subsequent dose of 300 mg of thehumanized immunoglobulin or antigen-binding fragment thereof as anintravenous infusion at about two weeks after the initial dose; (c)followed beginning at week six by a third and subsequent doses of 165 mgof the humanized immunoglobulin or antigen-binding fragment thereof as asubcutaneous injection every week, every two weeks or every three weeksas needed; wherein the dosing regimen induces a clinical response andclinical remission in the inflammatory bowel disease of the patient; andfurther wherein the humanized immunoglobulin or antigen-binding fragmenthas binding specificity for the α4β7 complex, wherein theantigen-binding region comprises three complementarity determiningregions (CDR1, CDR2, and CDR3) of a light chain variable region andthree complementarity determining regions (CDR1, CDR2, and CDR3) of aheavy chain variable region of the amino acid sequence set forth below:light chain: CDR1 SEQ ID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13;heavy chain: CDR1 SEQ ID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

In another aspect, the invention relates to a dosing regimen for thetherapeutic treatment of inflammatory bowel disease, wherein the dosingregimen comprises the step of: administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to a subcutaneous or intramuscular dosing regimenwhich maintains a mean steady state serum trough concentration of about9 to about 13 μg/mL of the antibody or antigen-binding fragment thereof;wherein the dosing regimen induces a clinical response and clinicalremission in the inflammatory bowel disease of the patient; and furtherwherein the humanized immunoglobulin or antigen-binding fragment hasbinding specificity for the α4β7 complex, wherein the antigen-bindingregion comprises three complementarity determining regions (CDR1, CDR2,and CDR3) of a light chain variable region and three complementaritydetermining regions (CDR1, CDR2, and CDR3) of a heavy chain variableregion of the amino acid sequence set forth below: light chain: CDR1 SEQID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

In another aspect, the invention relates to a dosing regimen for thetherapeutic treatment of inflammatory bowel disease, wherein the dosingregimen comprises the step of: administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to a subcutaneous or intramuscular dosing regimenwhich maintains a steady state serum trough concentrations of about 35to about 40 μg/mL of the antibody or antigen-binding fragment thereof;wherein the dosing regimen induces a clinical response and clinicalremission in the inflammatory bowel disease of the patient; and furtherwherein the humanized immunoglobulin or antigen-binding fragment hasbinding specificity for the α4β7 complex, wherein the antigen-bindingregion comprises three complementarity determining regions (CDR1, CDR2,and CDR3) of a light chain variable region and three complementaritydetermining regions (CDR1, CDR2, and CDR3) of a heavy chain variableregion of the amino acid sequence set forth below: light chain: CDR1 SEQID NO: 11, CDR2 SEQ ID NO: 12, CDR3 SEQ ID NO: 13; heavy chain: CDR1 SEQID NO: 8, CDR2 SEQ ID NO: 9, CDR3 SEQ ID NO: 10.

In some embodiments, the method of treatment, dose or dosing regimenreduces the likelihood that a patient will develop a HAHA response tothe anti-α4β7 antibody. The development of HAHA, e.g., as measured byantibodies reactive to the anti-α4β7 antibody, can increase theclearance of the anti-α4β7 antibody, e.g., reduce the serumconcentration of the anti-α4β7 antibody, e.g., lowering the number ofanti-α4β7 antibody bound to α4β7 integrin, thus making the treatmentless effective. In some embodiments, to prevent HAHA, the patient can betreated with an induction regimen followed by a maintenance regimen. Insome embodiments, there is no break between the induction regimen andthe maintenance regimen. In some embodiments, the induction regimencomprises administering a plurality of doses of anti-α4β7 antibody tothe patient. To prevent HAHA, the patient can be treated with a highinitial dose, e.g., at least 1.5 mg/kg, at least 2 mg/kg, at least 2.5mg/kg, at least 3 mg/kg, at least 5 mg/kg, at least 8 mg/kg, at least 10mg/kg or about 2 to about 6 mg/kg, or frequent initial administrations,e.g., about once per week, about once every two weeks or about onceevery three weeks, of the standard dose when beginning therapy with ananti-α4β7 antibody. In some embodiments, the method of treatmentmaintains at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or at least 95% of patients asHAHA-negative. In other embodiments, the method of treatment maintainspatients as HAHA-negative for at least 6 weeks, at least 10 weeks atleast 15 weeks, at least six months, at least 1 year, at least 2 years,or for the duration of therapy. In some embodiments, the patients, or atleast 30%, at least 40%, at least 50% or at least 60% of patients whodevelop HAHA maintain a low titer, e.g., ≤125, of anti-α4β7 antibody. Inan embodiment, the method of treatment maintains at least 70% ofpatients as HAHA-negative for at least 12 weeks after beginning therapywith an anti-α4β7 antibody.

The formulation may be administered to an individual (e.g., a human)alone or in conjunction with another agent. A formulation of theinvention can be administered before, along with or subsequent toadministration of the additional agent. In one embodiment, more than oneformulation which inhibits the binding of α4β7 integrin to its ligandsis administered. In such an embodiment, an agent, e.g., a monoclonalantibody, such as an anti-MAdCAM or an anti-VCAM-1 monoclonal antibodycan be administered. In another embodiment, the additional agentinhibits the binding of leukocytes to an endothelial ligand in a pathwaydifferent from the α4β7 pathway. Such an agent can inhibit the binding,e.g. of chemokine (C-C motif) receptor 9 (CCR9)-expressing lymphocytesto thymus expressed chemokine (TECK or CCL25) or an agent which preventsthe binding of LFA-1 to intercellular adhesion molecule (ICAM). Forexample, an anti-TECK or anti-CCR9 antibody or a small molecule CCR9inhibitor, such as inhibitors disclosed in PCT publication WO03/099773or WO04/046092, or anti-ICAM-1 antibody or an oligonucleotide whichprevents expression of ICAM, is administered in addition to aformulation of the present invention. In yet another embodiment, anadditional active ingredient (e.g., an anti-inflammatory compound, suchas sulfasalazine, azathioprine, 6-mercaptopurine, 5-aminosalicylic acidcontaining anti-inflammatories, another non-steroidal anti-inflammatorycompound, a steroidal anti-inflammatory compound, or antibioticscommonly administered for control of IBD (e.g. ciprofloxacin,metronidazole), or another biologic agent (e.g. TNF alpha antagonists)can be administered in conjunction with a formulation of the presentinvention.

In an embodiment, the dose of the co-administered medication can bedecreased over time during the period of treatment by the formulationcomprising the anti-α4β7 antibody. For example, a patient being treatedwith a steroid (e.g. prednisone, prednisolone) at the beginning, orprior to, treating with the anti-α4β7 antibody formulation would undergoa regimen of decreasing doses of steroid beginning as early as 6 weeksof treatment with the anti-α4β7 antibody formulation. The steroid dosewill be reduced by about 25% within 4-8 weeks of initiating tapering, by50% at about 8-12 weeks and 75% at about 12-16 weeks of tapering duringtreatment with the anti-α4β7 antibody formulation. In one aspect, byabout 16-24 weeks of treatment with the anti-α4β7 antibody formulation,the steroid dose can be eliminated. In another example, a patient beingtreated with an anti-inflammatory compound, such as 6-mercaptopurine atthe beginning, or prior to, treating with the anti-α4β7 antibodyformulation would undergo a regimen of decreasing doses ofanti-inflammatory compound similar to the tapering regimen for steroiddosing as noted above.

In one embodiment, the method comprises subcutaneously administering orintramuscularly administering an effective amount of a formulation ofthe invention to a patient. In another embodiment, the formulation canbe prepared for self-administration.

If the formulation is in a solid, e.g., dry state, the process ofadministration can comprise a step of converting the formulation to aliquid state. In one aspect, a dry formulation can be reconstituted,e.g., by a liquid as described above, for use in injection, e.g.intravenous, intramuscular or subcutaneous injection. In another aspect,a solid or dry formulation can be administered topically, e.g., in apatch, cream, aerosol or suppository.

The invention also relates to a method for treating a disease associatedwith leukocyte infiltration of tissues expressing the molecule MAdCAM(e.g., MAdCAM-1). The method comprises administering to a patient inneed thereof an effective amount of an anti-α4β7 antibody formulation ofthe invention. In an embodiment, the disease is graft versus hostdisease. In some embodiments, the disease is a disease associated withleukocyte infiltration of tissues as a result of binding of leukocytesexpressing α4β7 integrin to gut-associated endothelium expressing themolecule MAdCAM (e.g., MAdCAM-1). In other embodiments, the disease isgastritis (e.g., eosinophilic gastritis or autoimmune gastritis),pancreatitis, or insulin-dependent diabetes mellitus. In yet otherembodiments, the disease is cholecystitis, cholangitis, orpericholangitis.

The invention also relates to a method for treating inflammatory boweldisease in a patient. In one embodiment, the method comprisessubcutaneously administering to the patient an effective amount of ananti-α4β7 antibody formulation of the invention. In some embodiments,the inflammatory bowel disease is ulcerative colitis or Crohn's disease.In other embodiments, the inflammatory bowel disease is Celiac disease,enteropathy associated with seronegative arthropathies, microscopic orcollagenous colitis, gastroenteritis (e.g., eosinophilicgastroenteritis), or pouchitis.

In some embodiments, treatment with an anti-α4β7 antibody does not alterthe ratio of CD4:CD8 lymphocytes. CD4:CD8 ratios can be measured inblood, lymph node aspirate, and cerebro-spinal fluid (CSF). The CSFCD4+:CD8+ lymphocyte ratios in healthy individuals are typically greaterthan or equal to about 1. (Svenningsson et al., J. Neuroimmunol. 1995;63:39-46; Svenningsson et al., Ann Neurol. 1993; 34:155-161). Animmunomodulator can alter the CD4:CD8 ratio to less than 1.

Articles of Manufacture

In another aspect, the invention is an article of manufacture whichcontains the pharmaceutical formulation of the present invention andprovides instructions for its use. The article of manufacture comprisesa container. Suitable containers include, for example, bottles, vials(e.g., dual chamber vials, a vial of liquid formulation with or withouta needle, a vial of solid formulation with or without a vial ofreconstitution liquid with or without a needle), syringes (such as dualchamber syringes, preloaded syringes, an auto-injector), cartridges, andtest tubes. The container may be formed from a variety of materials suchas glass, metal or plastic. The container holds the formulation and alabel on, or associated with, the container may indicate directions foruse. In another embodiment, the formulation can be prepared forself-administration and/or contain instructions for self-administration.In one aspect, the container holding the formulation may be a single-usevial. In another aspect, the container holding the formulation may be amulti-use vial, which allows for repeat administration (e.g., from 2-6administrations) of the formulation, e.g., using more than one portionof a reconstituted formulation. The article of manufacture may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes andpackage inserts with instructions for use as noted in the previoussection.

In one embodiment, the article of manufacture is a syringe with aneedle. The gauge of the needle may be 25 G, 26 G, 27 G, 29 G, 30 G. Athin wall needle, e.g, 19 G or 23 G, or greater, can facilitateinjection of a high viscosity formulation. In one aspect, the needlegauge is 27 G or greater. Needle length can be suitable for subcutaneousadministration, and can be about ½ inch, about ⅝ inch or 1 inch long. Insome embodiments, the syringe is a pre-filled syringe.

Pre-Filled Syringe Product Development

In some aspects, there are several product attributes that are desiredfor a protein product (e.g., an anti-α4β7 antibody) in a pre-filledsyringe (PFS) (e.g., for use in administration of a formulation forsubcutaneous or intramuscular delivery). It is helpful to balance someof the attributes to mitigate competing effects. For example, when a lowinjection volume is desired, a high protein concentration for theformulation may be preferred. However, in the case of a high proteinconcentration, there can be higher rates of impurity formation (e.g.,aggregated impurities that leach into formulation from syringe) andhigher manual forces needed to operate the syringe. A small needle sizeused for patient comfort at the injection site, may require high forcesto operate the syringe. An understanding of how product stability andperformance is affected by both formulation and syringe parameters suchas protein concentration, pH, and needle inner diameter aids in thedevelopment of a protein product (e.g., an anti-α4β7 antibody in apre-filled syringe).

In one aspect, a method of developing a protein product (e.g., ananti-α4β7 antibody) for use in a pre-filled syringe comprises varyingsyringe parameters and formulation parameters together, e.g., in acoordinate fashion or simultaneously. This can lead to a betterunderstanding of the range of protein stability and product performancethat can be expected from a protein product in a pre-filled syringe thanif each aspect is varied separately or in series.

The development of a pre-filled syringe product (e.g., an anti-α4β7antibody) relates to understanding that at some point, there is a liquidformulation in contact with several components of the pre-filled syringe(FIG. 15). For example, the formulation can be in contact with a syringebarrel, which can be constructed of glass (e.g., type I borosilicateglass) or plastic (e.g., cyclic olefin polymer (COP), cyclic olefincopolymer (COC), polypropylene or polytetrafluoroethylene). Theformulation can be in contact with the syringe, plunger and/or tip cap,which can be elastomeric (e.g., of the same or different materials(e.g., plastic, such as polyethylene, polystyrene or polypropylene orelastic, such as rubber (natural, synthetic, butyl) or silicone)). Theformulation may be in contact with a lubricant that is added to an innersurface of the barrel for ease of plunger movement. The lubricant canbe, for example, silicone oil, mineral oil or glycerin. In theembodiment of a staked needle syringe, there can be a metal alloy needle(e.g., stainless steel needle and adhesive used to glue the needle inplace). A consideration for a protein product in a pre-filled syringe isthat the liquid protein solution is in direct contact with one or moreof these syringe components throughout the shelf life of the product.Both the formulation and syringe components can have an impact on thestability of the product.

Formulation parameters that can affect pre-filled syringe productstability include protein concentration, pH, buffer type, bufferconcentration, ionic strength, stabilizer type, and stabilizerconcentration. Examples of stabilizers for protein formulations include,for example, ionic salts, polysaccharides, amino acids, antioxidants,chelators, and surfactants as described in earlier sections.

Syringe components that can affect pre-filled syringe product stabilityinclude, for example, lubricant, composition of plunger and tip cap, andimpurities. The amount of lubricant (e.g., silicone oil on the syringebarrel) may affect product stability. The composition of the plunger andtip cap, which can affect oxygen permeability of these components andintroduce leachables from these components into the protein product(e.g., the anti-α4β7 antibody formulation) may also affect productstability. Another syringe parameter that can affect product stabilityincludes the type and/or amount of impurities (e.g., heavy metal (e.g.,tungsten)) that can leach into the product formulation (e.g., from inthe barrel (e.g., glass barrel) and/or needle (e.g., stainless steelneedle)). (See also Ludwig et al. J. Pharm. Sci. 99:1721-1733 (2010);Nashed-Samuel et al., American Pharmaceutical Review January/February:74-80 (2011); Badkar et al. AAPS PharmSciTech 12:564-572)

A pre-filled syringe can be injected manually or used with anauto-injector device. Functional testing of the pre-filled syringeincludes measuring the break-loose force, the force required to beginmovement of the plunger, and the gliding force, the force needed toinject the contents of the syringe at a constant rate. The mechanicalperformance of the pre-filled syringe can be dependent on severalformulation and syringe parameters such as the viscosity of theformulation and the amount of lubricant (e.g., silicone oil) in thesyringe.

Several attributes of protein products in pre-filled syringes andformulation or syringe factors that can impact those product attributesare shown in Table 1. Many product attributes can be a complex functionof several formulation and syringe parameters. For example, syringegliding force is a function of formulation viscosity, although viscositycan be dependent on several formulation factors, such as proteinconcentration, stabilizer concentrations, and pH.

TABLE 1 Product Attributes for Protein Products in Pre-filled Syringesand the Potential Formulation and Syringe Parameters that may ImpactThese Attributes Protein Formulation Syringe Parameters that ProductParameters that may may Impact Product Attribute Impact ProductAttribute Attribute Osmolality Stabilizer concentrations, None pH,protein concentration Viscosity Stabilizer concentrations, None pH,protein concentration Syringe Break Viscosity, protein Injection speed,needle Loose concentration, surfactant length, needle ID, syringe andconcentration barrel ID, silicone oil Gliding Force amount, plungerformulation and shape Rate of Protein Stabilizer concentrations, NoneDeamidation pH, protein concentration Rate of ProteinStabilizer/antioxidant Plunger and tip cap Oxidation concentrations, pH,protein formulation (oxygen concentration, surfactant permeability),heavy metal concentration, dissolved impurity levels, size of air oxygenbubble Rate of Soluble Stabilizer concentrations, Silicone oil amount,heavy pH, Aggregate protein concentration, metal impurity levels, sizeof Formation surfactant concentration, air bubble dissolved oxygen insolution Rate of Sub- Stabilizer concentrations, Silicone oil amount,Heavy visible pH, metal impurity levels,, size and Visible proteinconcentration, of air bubble, syringe Proteinaceous surfactantconcentration internal surface area Particulate Formation

In one aspect, a surfactant, such as polysorbate 20 or polysorbate 80can be added to protein formulations in pre-filled syringes (e.g., toprevent protein molecules from adsorbing and denaturing at theliquid/air and/or liquid/lubricant (e.g., silicone oil) interfaces).Surface adsorption and denaturation of protein molecules can be onemechanism for the nucleation of sub-visible and visible proteinaceousparticles. Addition of a surfactant to a pre-filled syringe, therefore,can reduce the formation of sub-visible and visible particles inpre-filled syringe products. In one embodiment, a small amount ofsurfactant can emulsify lubricant (e.g., silicone oil droplets in thesolution and thereby reduce the formation of subvisible and visiblelubricant (e.g., silicone oil droplets)) (Ludwig et al., supra). Inanother embodiment, the amount of surfactant in a formulation isminimized, due to potential harmful effects of high amounts ofsurfactants on protein formulations. Peroxide impurities present inpolysorbates can lead to increased protein oxidation (Wang and Wang J.Pharm. Sci. 91:2252-2264 (2002)). High amounts of surfactant canemulsify a significant amount of silicone oil from the walls of thesyringe and lead to an increase in the functional gliding force over theshelf life. Product development studies should be designed to examinethe effect of varying surfactant levels on both product stability andsyringe performance.

The complex interactions between the formulation and the syringeparameters in protein/PFS systems are amenable to examination of thesesystems using a Quality by Design (QbD) or Design of Experiments (DOE)approach. Studies can be designed that simultaneously vary formulationand syringe parameters to gain a better understanding of these complexsystems. This results in a comprehensive approach to the development ofpre-filled syringe products. Table 2 shows an example of the inputparameters and levels that may go into a design of experiment for apre-filled syringe product and an example of the analytical testing tobe employed. Depending on the type of experimental design that is usedfor QbD study, the number of experiments could vary from 9 for ascreening design to 81 for a full-factorial design (all possiblecombinations). The higher the number of experiments, the higher thenumber of interactions between product parameters that can be resolved.Software designed for this analysis, for example, JMP® statisticaldiscovery software (Cary, N.C.), can be helpful for QbD studies. Thisanalysis results in a quantitative understanding of how formulation andsyringe parameters interact to impact product attributes.

TABLE 2 Example of an Experimental Design for a Liquid Protein Productin a Pre-filled Syringe Formulation Input Parameters Levels AnalyticalTesting Protein 50, 100, 150 Protein stability by Concentration mg/mLsize exclusion pH 5.5, 6.5, 7.5 chromatography and ion exchangechromatography Surfactant 0.01, 0.08, 0.15 Break loose and glidingConcentration % force over time using force testing Amount of 0.2, 0.5,0.8 Formation of sub-visible Silicone Oil in mg/syringe proteinaceousSyringe particles and silicone oil droplets over time using microflowimaging or/coulter counter

An example of a predictive model that can be obtained from the exampleexperiment shown in Table 2 is given below, where C_(n) are numericalconstants.

Soluble Aggregate Formation over Time=C₀+C₁[ProteinConcentration]+C₂[Protein Concentration]²+C₃[pH]+C₄[SurfactantConcentration]+C₅[Lubricant Amount]

Numerous syringe parameters can affect product stability andperformance, therefore an embodiment includes characterization of howthe allowable tolerances in syringe parameters affect product stabilityand performance. The amount of lubricant (e.g., silicone oil) on thesyringe barrel may vary 50-100% from syringe to syringe. This variationin the amount may affect several product characteristics as shown inTable 1. The inner diameter of the syringe barrel can vary from syringeto syringe which affects injection forces. For staked-needle syringes,the needle inner diameter may vary from lot to lot or from manufacturerto manufacturer, which will affect injection forces. By using a QbDapproach to examine how syringe parameters affect performance,predictive models can be obtained that can be used to estimate how theallowable tolerances in syringe parameters may affect productperformance. Predictive models that are obtained using a QbD approachcan be used to select formulation and syringe parameters that meetdesired product attributes and to predict product stability andperformance.

The pre-filled syringe may contain an addition of silicone emulsion ortungsten to the protein formulation. Exemplary amounts of silicone thatmay be present in the pre-filled syringe range from about 0.3 mg toabout 0.8 mg. In one aspect, the amount of silicone that may be presentin the pre-filled syringe is about 0.3 mg, about 0.4 mg, about 0.5 mg,about 0.6 mg, about 0.7 mg, or about 0.8 mg. In another aspect, theviscosity of the formulation will range from 2 to 60 cP, resulting ininjection forces of 5N to 80N at a speed of 200 mm/min. In still yetanother aspect, the viscosity of the formulation will range from 4 to 27cP resulting in injection forces of 10 N to 40 N at a speed of 200mm/min.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areincorporated herein by reference.

Protocol for Making Formulation

A solution of anti-α4β7 antibody is diafiltered in a tangential flowfiltration system to reach a specified concentration in citrate,histidine, arginine buffer, then pooled and mixed with a solution ofpolysorbate 80 in citrate, histidine, arginine buffer. The solution isstored at −70° C. in either 2 L or 5 L bottles. The solution is thenthawed and filtered twice through a 0.2 μm filter. Approximately 1.0 mLis filled into a sterilized syringe and closed with a sterilized plunger(stopper). The formulation is stored and the final drug product isshipped in syringes at 2-8° C.

EXAMPLES Example 1

Formulation Manufacturing

Factors

Excipient Concentrations

The formation of aggregates in the antibody formulation was tested. AnSEC aggregates model was developed from experimental data that examinedprotein concentration, pH, and surfactant:protein molar ratio. At a pHrange from 6.0 to 6.5, the formation of aggregates was similar with thepolysorbate 80 to protein molar ratio range from 0.7 to 1.5. (FIG. 6)Generally, at PS80:Protein ratios greater than 1.5, the aggregatesformation rate increases with increasing pH. (FIG. 7)

An experiment examining the formation of SEC aggregates in the presenceof air was performed. Eleven different formulations of varyingcomposition were put into borosilicate vials and capped with elastomericstoppers with an air headspace. An identical set of formulations werecreated, and the air headspace was displaced with argon. These sampleswere placed on stability at 40° C. for two weeks. All the samples withthe air headspace resulted in large amounts of aggregates at the end ofthe experiment in comparison to the same formulation with the argonheadspace.

TABLE 3 Aggregates Aggregates Protein PS Air Argon Conc. SucroseHistidine Arginine 80 Samples Samples Sample (mg/ml) (%) (mM) (mM) (%)pH (%) (%) 1 60 2 25 75 0.05 6.2 0.64 0.48 2 60 4 25 75 0.05 7 0.62 0.423 160 4 50 75 0.14 6.2 0.92 0.73 4 160 2 50 75 0.14 7 1.16 0.74 5 60 250 125 0.05 7 1.28 0.33 6 60 4 50 125 0.05 6.6 0.48 0.36 7 160 4 25 1250.14 7 1.04 0.70 8 160 2 25 125 0.14 6.2 1.06 0.75 9 160 3 25 75 0.146.6 1.09 0.78 10 110 3 50 125 0.10 6.2 0.65 0.47 11 110 2 25 75 0.10 6.60.90 0.62

Based on these experiments, SEC aggregates were hypothesized to form byoxidation or by disulfide bond formation. The addition of antioxidantsand/or chelators was explored. A formulation containing 40 mM Histidine,90 mM Arginine, and 160 mg/mL protein with a polysorbate 80 to proteinmolar ratio of 1.5 at pH 6.6 was made. To the formulation, 25 mMcitrate, 5 mM citrate, 5 mM EDTA, 25 mM cysteine, or 5 mM cysteine wasadded. All 3 additional excipients reduced the formation of aggregates(FIG. 8). The addition of the antioxidants and/or chelators were rankedin order of performance as citrate>EDTA>cysteine. Either 5 or 25 mMcitrate reduced the formation of SEC aggregates as compared to thecontrol formulation.

An experiment was performed to determine the effects of pH, proteinconcentration, citrate concentration, histidine concentration and thepolysorbate 80 to protein molar ratio. The pH was varied from 6.0 to6.3, the protein concentration was varied from 60 to 160 mg/mL, thecitrate concentration was varied from 0 to 25 mM, the histidineconcentration was varied from 25 to 50 mM, and the polysorbate 80 toprotein molar ratio was varied from 0.7 to 1.5. Formulations were filledin 1 ml long, 27 G½″ syringes (0.55+/−0.2 mg silicone). All formulationscontained approximately 125 mM arginine.

Stability was tested at 40° C. for two weeks, using CEX and SEC. Theresults (FIG. 9 and Table 4) show a reduction in aggregate formationwith the presence of 25 mM citrate in the formulation, while increasingthe protein concentration increased the rate of aggregate formation. Theamount of monomer shows opposite trends to the aggregate formation at25° C. and 40° C., while at 5° C. the amount of monomer is essentiallyunchanged for up to 24 months (Table 5).

Another set of formulations explored the rate of formation of SECaggregates in the presence of 40-63 mM citrate but with no histidine at40° C., 25° C., 5° C. The rate of aggregate formation in theseformulations was slightly higher than formulations with histidine at 40°C. However, at 5° C., the rate of formation of aggregates in theformulations with citrate and no histidine were comparable to theformulations containing citrate and histidine (Table 6). Also at 5° C.,the amount of monomer is essentially unchanged for up to 24 months(Table 7).

TABLE 4 Change Change Change

in in in Change

in

in

 After

 After

 After

Protein

Citrate Arginine

of 12 24 12

After

Conc. Conc. Conc. Conc.

1 

(mg/mL) pH (mM) (mM) (mM)

at 5° C. at 5° C. at 25° C. at

° C. 1  62 6.4 50 25 125 0. 

0.4 01 0.1 0. 

0.2 2  60 6.4 50 0 125 1. 

0.4 0 

1.1 1.3 0.4 3 1 

6.4 50 25 125 1. 

0.4 02 0. 

1.3 0.5 4 161 6.3

 0 0 125 0.7 0.4 06 0.7 2. 

0.3 5  60 6.2

 0 25 125 1. 

0.4 02 0.2 0. 

0.2 6 110 6.0 50 0 125 0. 

0.4 04 0.6 1.7 0. 

7 162 6.2 50 25 125 0. 

0.4 03 0.3 1.1 0. 

8 160 6.0 50 0 125 1. 

0.4 04 0.6 2.2 0.9 9 169 6.3 25 25 125 0.7 0. 

— 0.3 — 0.6 10 1 

6.3 25 25 12 

1.0 0. 

— — — 0.6

indicates data missing or illegible when filed

TABLE 5

Change Change Change Change

Initial in in in in

on

Protein

Citrate Arginine

of After 12 After 24 After 12

After

Conc. Conc. Conc. Conc.

1 

(mg/mL) pH (mM) (mM) (mM)

(%) 5° C. at 5° C. 25° C. at 40° C. 1  62 6.4 50 25 125 0.7

0.4 0.1 −2.5 −1.3 2  60 6.4 50 0 125 1.5

0.2 −1.0

−2.0 3 1 

6.4 50 25 125 1.5

0. 

0.0

4 161 6.3 50 0 125 0.7

0.0 −0.4

−2.1 5  60 6.2 50 25 125 1.5

0.3 0.2 −2.2 −1.4 6 110 6.0 50 0 125 0.7

0.1 −0.3 −3.6 −2.1 7 162 6.2 50 25 125 0.7

0.2 −0.1 −2. 

−1.7 8 160 6.0 50 0 125 1.5

0.1 −0.4 −4.2 −2.3 9 169 6.3 25 25 125 0.7

— −0.1 —

10 1 

6.3 25 25 12 

1.0

— — — −1.7

indicates data missing or illegible when filed

TABLE 6 Initial Change in Change in Change in Change in

Amount

Protein

Citrate Arginine

of After 12 After 24 After 12 After 1

Conc. Conc. Conc. Conc.

# (mg/mL) pH (mM) (mM) (mM)

(%) 5° C. 5° C. 25° C. 40° C. 11 160 6.3 0 40 125 0.7 0. 

— 0.4 — 0.9 12 165 6.3 0 40 125 1.5 0.6 0.3 — — 0.9 13  62 6.2 0 40 1251.5 0. 

— — 1.7 0.4 14 170 6.1 0 40 125 1.5 0. 

— — — 0.9 15 165 6.5 0 63 125 1.5 0. 

— — — 1.0 16 160 6.3 0 40 125 1.0 0.6 0.6 — — 0.9

indicates data missing or illegible when filed

TABLE 7 Initial Change in Change in Change in Change in

Amount

Protein

Citrate Arginine

of After 12 After 24 After 12 After 1

Conc. Conc. Conc. Conc.

# (mg/mL) pH (mM) (mM) (mM)

(%) 5° C. 5° C. 25° C. 40° C. 11 160 6.3 0 40 125 0.7

— −0.3 — −22 12 165 6.3 0 40 125 1.5

0.1 — −3.4 −21 13  62 6.2 0 40 125 1.5

— — — −14 14 170 6.1 0 40 125 1.5

— — — −20 15 165 6.5 0 63 125 1.5

— — — −23 16 160 6.3 0 40 125 1.0

0.1 — — −20 pH

indicates data missing or illegible when filed

Several pH experiments were done to determine the effects of pH on CEXdegradation at 5° C. The vedolizumab antibody formulation comprised 160mg/ml of anti-α4β7 antibody, 125 mM arginine, 50 mM histidine, and 25 mMcitrate. Several different pH levels, 6.3, 6.5, 6.7 and 6.9 were testedfor stability at 40° C., 25° C., and 5° C.

The CEX models at 40° C. show (FIG. 10) that pH influences CEXdegradation the most. The pH of formulations containing histidinedecreases with increasing temperature, however the pH of citrateformulations was shown to not be affected by temperature (FIG. 11). Thehistidine/citrate formulation was determined to have good stability at apH of 6.8 at 40° C. after 1 week, 6.3-6.5 at 25° C. after 6 months and6.3-6.5 at 5° C. after 6 months. Based on additional studies, thestability of the formulations were similar at 25° C. and 5° C. for thepH range of 6.2 to 6.9 (Tables 8 and 9).

TABLE 8 Difference in % Relative Area Over Time at

 5° C.

Change in Change in Change in Change in Change in Change in

in CEX in CEX in CEX in CEX in CEX in CEX Protein

Citrate Arginine

Acidic Acidic Basic Basic Major Major Conc. Conc. Conc. Conc.

After 6 After 12 After 6 After 12 After 6 After 12 (mg/mL) pH (mM) (mM)(mM)

(%) (%) (%) Months Months Months Months Months Months 157 6.4 30 25 1251.5

6.8

 8.5 17.4 7.1 3.4 −15.6 −20.8 162 6.2 30 25 125 0.7

6.9

 4.4

−15.2 −21.4 158 6.3 30 25 125 1.5

5.5

 2.2 — 4.9 — −14.4 — 160 6.4 42 25 125 1.5

5.5

 9.4 — 1.0 — −14.4 — 147

25 125 2.1

14.8 — 3.6 — −16.5 — 147 6.9

25 125 2.2

14.5 — 3.2 — −17.9 — 153 6.7

25 125 1.5

— 0.3 — −17.3 — 154 6.9

25 125 1.5

— 0.5 —

— 170 6.5 30 25 125 1.0

4.6

— 4.4 — −15.2 — 170 6.5 30 25 125 1.5

4.6

11.1 — 5.2 — −16.4 — 160 6.5 30 25 125 1.5

11.8 — −2.6 — −11.9 —

indicates data missing or illegible when filed

TABLE 9 Difference in % Relative Area Over Time at 5° C. Change ChangeChange Change Change Change PS80: in CEX in CEX in CEX in CEX in CEX inCEX Protein

Citrate Arginine Protein Acidic Acidic Basic Basic Major Major Conc.Conc. Conc. Conc.

After 6 After 24 After 6 After 24 After 6 After 24 (mg/mL) pH (mM) (mM)(mM) Ratio Months Months Months Months Months Months 157 6.4 50 25 1251.5 0.2 0.6 2.3 −0.9 −2.5 0.3 162 6.2 50 25 125 0.7 −0.2 −0.7 4.3 2.2−4.1 −1.5 158 6.3 50 25 125 1.5 0.0 — 1.9 — −1.9 — 160 6.4 42 25 125 1.50.1 — 1.3 — −1.8 — 147 6.7 45 25 125 2.1 1.7 — 1.3 — −3.4 — 147 6.9 4525 125 2.2 1.6 — 1.1 — −2.7 — 153 6.7 46 25 125 1.5 1.7 — 0.4 — −2.1 —154 6.9 46 25 125 1.5 2.1 — 0.4 — −2.4 — 170 6.5 50 25 125 1.0 0.9 — 1.6— −2.5 — 170 6.5 50 25 125 1.5 0.8 — 1.6 — −2.5 — 160 6.5 50 25 125 1.511.8 — −2.3 — −11.9 —

indicates data missing or illegible when filed

Example 2

Stability

Four different anti-α4β7 antibody formulations were tested for stabilityover the course of twelve months. Formulations having a pH of 6.0-6.2showed approximately 1-2% less major species than formulations having apH of 6.3-6.4 (FIG. 12). Formulations having a pH of 6.3-6.4 showed lessthan 1% change in basic or major species at 5° C.

Ten different anti-α4β7 antibody formulations were tested for stabilityby SEC over the course of twelve months (Table 10). The formulationswith 60 mg/mL protein concentration and containing 25 mM citrate had achange in aggregates of 0.1-0.2% after 1 year, while formulationscontaining 160 mg/mL protein and 25 mM citrate had an increase ofaggregates from 0.2-0.3% over 1 year. There was an increase of 0.4-0.6%aggregates for formulations containing 60, 110, or 160 mg/mL proteinwith no citrate.

TABLE 10 Change in % Aggregates Protein Histidine Citrate Arginine PS80at 5° C. Formulation Concentration Concentration ConcentrationConcentration Molar After 1 # (mg/mL) pH (mM) (mM) (mM) Ratio Year 1 626.41 50 25 125 0.7 0.11 2 60 6.35 50 0 125 1.5 0.50 3 157 6.44 50 25 1251.5 0.23 4 161 6.3 50 0 125 0.7 0.56 5 60 6.19 50 25 125 1.5 0.16 6 1106.03 50 0 125 0.7 0.39 7 162 6.19 50 25 125 0.7 0.26 8 160 6 50 0 1251.5 0.44 9 165 6.28 0 40 125 1.5 0.30 10 160 6.3 0 40 125 1.0 0.33

Example 3

Viscosity

The injection force needed to administer the pharmaceutical formulationis related to the viscosity of the formulation. Formulations withvarying pH and varying concentrations of protein, arginine, histidine,citrate, sucrose, and polysorbate 80 were made. The viscosity of theseformulations was tested. A statistical model of the Ln (viscosity) wasdeveloped. The model showed that the viscosity is affected mainly byprotein concentration and pH (FIG. 13). Sucrose, histidine and argininealso can have a minor effect on viscosity. In some protein formulations,sodium chloride is added to reduce the viscosity of the formulation. Itis known, however, that the effect of sodium chloride on viscosity isprotein and formulation dependent.

Sodium chloride was added to a formulation containing 140 mg/mlvedolizumab, 125 mM arginine, 25 mM histidine, 25 mM citrate, andpolysorbate 80 at a 1.5 polysorbate 80 to protein molar ratio, and a pHof 6.4. The NaCl did not have any effect on the viscosity of theformulation.

The effects of the viscosity on the injection force of various syringestested are shown in FIGS. 16A and 16B.

Example 4

Methods

Cation Exchange Chromatography (CEX)

A phosphate/sodium chloride gradient on a weak cation exchange column isused in a high performance liquid chromatography system to separatecharged species in anti-α4β7 antibody formulations and determine thecharge composition of the antibody species. Acidic Isoforms elute beforethe Major Isoform and Basic Isoforms elute after the Major Isoform.

Stability data for a vedolizumab formulation generated using a CEX assayindicated that the % Major Isoform was above 55.0%.

Capillary Isoelectric Focusing (cIEF)

cIEF is performed using an iCE280 whole column detection cIEF system(Convergent Biosciences, Toronto, Ontario). Choice of ampholyte can beas recommended by the manufacturer or can be a combination ofcommercially available ampholytes. A useful combination is a mixture of3-10 and 5-8 PHARMALYTE™ (GE Healthcare, Piscataway, N.J.).

Stability data for a vedolizumab formulation generated using a cIEFassay indicated that the % Major Isoform was about 53%, the % AcidicSpecies was about 42% and the % Basic Species was about 5%.

Size Exclusion Chromatography (SEC)

SEC is performed using an analytical SEC column (Tosoh Bioscience, LLC,King of Prussia, Pa.). The mobile phase is a phosphate-buffered salinesolution and the absorbance is monitored at 280 nm.

Stability data for a vedolizumab formulation generated using an SECassay indicated that the % Monomer was 99.0%, the % Aggregates was <0.5%and the % Low Molecular Weight substances was <1.0%.

SDS-PAGE Assay

SDS-PAGE is performed using an Invitrogen (Carlsbad, Calif.)Tris-Glycine gel, 4-20% for reducing condition and 4-12% fornon-reducing condition. The reconstituted antibody formulation sample isdiluted in liquid formulation buffer then diluted one to two withTris-Glycine SDS Sample Buffer (2×, Invitrogen) either with 10%2-mercaptoethanol (reducing sample buffer) or without 2-mercaptoethanol(non-reducing sample buffer). Samples are briefly heated and loaded incomparison with a molecular weight marker (Invitrogen). The gels arestained with colloidal coomassie blue (Invitrogen) according to themanufacturer's instruction. Protein bands are analyzed by densitometryto identify the % heavy and light chain for reduced gels and % IgG fornon-reduced gels.

Binding Efficacy

HuT78 cells (human T cell lymphoma cells, American Type CultureCollection, Manassas, Va.) suspended in 1% BSA in PBS, 0.01% sodiumazide are contacted with serial dilutions of primary test antibody.After incubation on ice, the cells are washed and treated withfluorescently labeled secondary antibody. After a further wash, thecells are fixed and suspended in FACS reagent for analysis by flowcytometry (Becton Dickinson Franklin Lakes, N.J.); also see U.S. Pat.No. 7,147,851.

Moisture by Karl Fischer

The formulation is titrated with methanol for a coulometric Karl Fischermoisture determination.

Example 5

Effects of Silicone from Syringe Products Pre-Filled with Anti-α4β7Antibody Formulation

A subcutaneous formulation consisting of 60-160 mg/mL of anti-α4β7protein in a buffer containing L-Histidine, L-Arginine Hydrochloride,Citrate and Polysorbate 80 is used to study the effects of silicone onthe stability of the protein formulations and container/closureattributes. The study is performed with a 0.5 mL fill.

Parameters including the protein concentration, the polysorbate 80 toprotein molar ratio, and the amount of silicone that is sprayed onto thesyringe barrels are explored. The range of each of the input parametersis shown in Table 11.

TABLE 11 Input Parameter Ranges Parameters Low High Protein 100 160Concentration (mg/mL) Polysorbate 0 2 80:Protein Ratio Silicone Amount0.4 0.8 (mg)

A design of experiment is used to determine the set of formulations tostudy. A reasonable number of formulations range from 6 to 8formulations. An example of the formulations that are tested is shown inTable 12.

TABLE 12 Protein PS80 Conc. PS80:Protein Concentration Silicone Run(mg/mL) Ratio (%) Level (mg) 1 100 1 0.087 0.8 2 100 2 0.174 0.8 3 160 00 0.8 4 160 2 0.279 0.4 5 100 0 0 0.4 6 160 2 0.279 0.8 7 100 1 0.0870.4 8 160 0 0 0.4 9 100 0 0 0 10 100 2 0.174 0 11 160 0 0 0 12 160 20.279 0

Some controls may be added to the set of formulations and tested at afew select time points.

These formulations are placed on stability at several differenttemperatures (e.g., 5° C., 25° C./60% RH, 40° C./75% RH) and pulled atvarious time points (e.g., 0 week, 1 week, 2 weeks, 4 weeks, 8 weeks, 12weeks, 6 months, and 12 months) for testing. Controls are tested at 0weeks, 12 weeks, 6 months and 12 months.

The tests that are performed at each stability time pull include SEC,CEX, Instron, MFI, and Silicone Quantification. 1 syringe is tested forInstron, with the expelled material being used for SEC, CEX, injectionforce measurements and microflow imaging (MFI), and siliconequantification.

Example 6

Analysis of Pre-Filled Syringe Components Filled with Anti-α4β7 AntibodyFormulation

This study explored how various syringe manufacturers, plunger (stopper)elastomeric materials, and the amount of PS80 in the formulationaffected the mechanical properties of the system and the stability ofthe formulation.

A design of experiment was created exploring 3 different syringemanufacturers, 2 different plunger (stopper) material types, and 2different PS80 to protein molar ratios. The rest of the formulation waskept constant at 170 mg/mL of protein, 125 mM arginine, 50 mM histidine,25 mM citrate, and a pH of 6.5. The needle size on these pre-filledsyringes was 27 G ½′ or 29 G ½″ thin wall. The experiments performed aredetailed in Table 15.

The experimental design inputs for the active portion of the experimentare shown below in Table 13, while the constants are shown in Table 14.The experimental design was created utilizing the inputs shown in Table13.

The list of experiments is shown in Table 10.

TABLE 13 DOE variables and levels with active formulation VariableValues PS80:Protein 1.0 1.5 Molar Ratio Syringe A B C ManufacturerPlunger (Stopper) 4432 4023 Coated Type

TABLE 14 Constants for active formulation Constant Value ProteinConcentration (mg/mL) 170 Arginine Concentration (mM) 125 HistidineConcentration (mM) 50 Citrate Concentration (mM) 25 pH 6.5

TABLE 15 Experimental details Plunger Run # Syringe Type (Stopper) PS801 C 4432 1 2 B 4432 1 3 A 4023 1 4 C 4023 1 5 B 4023 1 6 A 4023 1.5 7 C4023 1.5 8 B 4023 1.5

A concentrated formulation anti-α4β7 formulation is spiked withpolysorbate 80 and diluted down to 170 mg/mL. The composition of thestarting formulation is shown below in Table 16.

TABLE 16 Starting Formulation buffer details Total Total Protein HisCitrate Arg (mg/ml) (mM) (mM) (mM) pH 183 50 25 125 6.48

For the dilution of the material to the desired formulation composition,stock solutions of PS80 in 25 mM Citrate, 50 mM Histidine, 125 mMArginine, pH 6.48 are made.

TABLE 17 Stock solution details Excipient Concentration PS 80 (%) 5

The dilution scheme for the formulations is detailed in Table 18.

TABLE 18 Dilution detail 50 nM Histidine, 125 mM Arginine, 25 nMStarting PS80 in Citrate Total Formulation His/Arg/Citrate pH 6.48Volume (uL) Buffer (uL) Buffer (uL) 27868.9 890.8 1240.3 30000.0 18579.2890.8 530.0 20000.0

Compounding is performed based on the dilution scheme, and the startingformulation should be weighed, while the other stock solutions can bepipetted volumetrically. Formulations are filtered. 0.5 mL offormulation is aliquotted into as many 1 mL Long syringes as possible.The syringes are stoppered by the stoppering machine with a 2-4 mmbubble. For each time point, there is one syringe stored needle down andone syringe stored sideways. The extra syringes are stored needle down.

The syringes are tested at 5, 25 and 40° C. on week 2 and at one month.Analytical testing (appearance, Instron, pH, osmolality, density,viscosity, SEC, CEX, and Brightwell) is performed initially and thenagain at 2 weeks at 25 and 40° C. and at 4 weeks at 25° C.

Example 7

Analysis of Subcutaneous Container Closures Used in 27 G Thin WallNeedle Syringes Pre-Filled with Anti-α4β7 Antibody Formulation

This study explores how various syringe models with a 27 G thin wallneedle and various plunger (stopper) manufacturers and models affect themechanical properties of the system and the stability of the formulationover time.

This study explores how the stability of the anti-α4β7 subcutaneousliquid formulation in a prefilled syringe and the mechanical propertiesof the syringe are affected by the syringe manufacturer and the plunger(stopper) model for syringes with a 27GTW needle. The data generatedfrom this study may determine the container/closure components for theliquid subcutaneous anti-α4β7 formulation.

The experimental design inputs are shown below in Table 19, while theconstants are shown in Table 20. The experimental design was createdutilizing the inputs shown in Table 19.

The list of experiments to be performed is shown in Table 21.

TABLE 19 Variable Values Syringe A B C Manufacturer Plunger (Stopper)4432 4023 Coated D E Type

TABLE 20 Constants for active formulation Constant Value ProteinConcentration (mg/mL) 160 Arginine Concentration (mM) 125 HistidineConcentration (mM) 50 Citrate Concentration (mM) 25 PS80 (%) 0.2 pH 6.5

TABLE 21 Experimental details Plunger Run # Syringe (Stopper) 1 B D 2 B4432 3 A 4432 4 B 4023 Coated 5 A D 6 C 4023 Coated 7 A 4023 Coated 8 CD 9 C 4432 10 C E

A concentrated anti-α4β7 formulation is spiked with polysorbate 80 anddiluted down to 160 mg/mL. The composition of the starting formulationis shown below in Table 22.

TABLE 22 Starting Formulation buffer details Total Total Protein HisCitrate Arg (mg/ml) (mM) (mM) (mM) pH 180 50 25 125 6.3

For the dilution of the material to the desired formulation composition,stock solutions of PS80 in 25 mM Citrate, 50 mM Histidine and 125 mMArginine, pH 6.3 are made.

TABLE 23 Stock solution details Excipient Concentration PS 80 (%) inHis/ 1.68 Arg/Citrate buffer pH 6.3

The dilution scheme for the formulations is detailed in Table 24.

TABLE 24 Dilution detail Starting Formulation in His Arg PS80 in TotalCitrate His/Arg/Citrate Volume buffer (mL) Buffer (mL) (mL) 78 10(1.68%) 88

Compounding is performed based on the dilution scheme, and the startingformulation should be weighed, while the other stock solutions can bepipetted volumetrically. Formulations are filtered. 0.5 mL offormulation is aliquotted into as many 1 mL Long syringes as possible.The syringes are stoppered by the stoppering machine with a 2-4 mmbubble. For each time point, there is one syringe stored needle down(horizontal position).

The syringes are tested at 5° C., 25° C./60% RH, and 40° C./75% RH at 1month, 3 months, 6 months, 9 months (optional), 12 months, 18 months and24 months.

The liquid formulations are analytically tested (concentration,osmolality, pH, Instron, MFI, SEC, and/or CEX) at 1, 3, 6, 9, 12, 18, 24month (5° C.); 1, 3, 6, 9, 12, 18, month (25° C.); 1, 3, 6, 9, 12, month(40° C.); and 1, 3, month (40° C.).

Example 8

Analysis of Subcutaneous Anti-α4β7 Antibody Formulation in PlasticPrefilled Syringes

This study is initiated to research the use of plastic syringes as thecontainer/closure system for an anti-α4β7 antibody subcutaneousformulation. The stability of a representative anti-α4β7 antibodysubcutaneous formulation in candidate plastic prefilled syringes isstudied. The data generated from this study helps to judge theapplicability of using a plastic syringe for a liquid subcutaneousanti-α4β7 antibody formulation.

Stability test samples are prepared as shown below. Stability tests areconducted under the storage conditions of 40° C./75% RH, 25° C./60% RH,and 5° C.

Two types of plastic syringes and one glass syringe (Control) in Table25 are tested with a liquid subcutaneous anti-α4β7 antibody formulationshown in Table 26. Table 27 shows the details of each set of samples tobe tested in the experiment.

TABLE 25 Plastic syringes Sample #1 Sample #2 Sample #3 Plastic syringePlastic syringe Glass syringe 1 2 (Control) Syringe Vendor F B AComponents Syringe: Syringe: Syringe: Glass polymer polymer Needle:Needle: Needle: 27G(TW) 27G(TW) 26G(RW) Rigid needle Rigid needle Luerlock tip shield shield cap Silicon coating Free Not free Not freePlunger Vendor F F ← Product 1 mL material A 1 mL material B ←description Silicon coating No Yes ←

TABLE 26 Anti-α4β7 antibody subcutaneous formulation (pH 6.5) ComponentComposition Anti-α4β7 antibody 160 mg/mL Arginine 125 mM Histidine 50 mMCitrate 25 mM PS80 (Protein Molar Ratio) 1.5 (0.2w/v %)

TABLE 27 Sample details PS 80 (MW: 1309.68) Plastic Protein Arg HisCitrate Protein syringe (MW: 150000) (MW: 174.20) (MW: 155.15) (MW:210.14) Molar Sample # vendor (mg/ml) (mM) (mM) (mM) (mM) (w/v %) RatiopH 1 F 160 1.067 125 50 25 0.21 1.5 6.5 2 B 160 1.067 125 50 25 0.21 1.56.5 3 (Cont.) A 160 1.067 125 50 25 0.21 1.5 6.5

Previously prepared liquid subcutaneous anti-α4β7 formulation are usedfor this investigation. Formulations are filtered. Sampling the filteredsolution for the quality test as “before filling” sample (Appearance,MFI, DLS). 0.5 mL of formulation are aliquotted into 1 mL plasticsyringes. The syringes are stoppered by the vacuum stoppering machine.The syringes are stored needle down.

An initial check is performed to measure pH, osmolality, density,viscosity, and protein concentration. Analytical testing (appearance,SEC (Aggregates, Monomer, LMW), CEX (Acidic, Main, Basic), glide force,MFI, DLS, and/or weight) is performed after 1 week, at 40° C., 2 weeks,40° C., 1 month, 5, 25 and 40° C., 3 months, 5 and 25° C., 6 months at 5and 25° C., 9 months at 5 and 25° C., and 12 months at 5 and 25° C.

Samples are taken at 1 month, 3 months, 6 months, 9 months and 12 monthsat 5° C. and 25° C. Samples are taken at 1 week, 2 weeks and 1 month at40° C.

Example 9

The samples were analyzed for appearance, injection force, SEC, CEX, andmicro-flow imaging at 5° C. and 25° C. at various time points that mayhave included 0, 1, 3, 6, and 12 months. The stability of theformulation as measured by SEC and CEX were similar to what wasdiscussed in Examples 1 and 2. For injection force testing, the glideforce were measured (Table 28). A statistical model determined that theonly significant factor affecting the glide force was the syringemanufacturer, where A had higher glide forces than B, which was greaterthan C (FIG. 17). The changes in glide force of the syringes over 12months at 5° C. and 6 months at 25° C. were less than 10 N, but mostlyless than 5 N.

TABLE 28 Plunger Syringe Needle (Stopper) PS80: Protein Initial GlideRun # Manufacturer Size Type Molar Ratio Force (N) 1 C 27G D 1 19.2 2 B27G D 1 22.9 3 A 29GTW E 1 25.0 4 C 27G E 1 18.5 5 B 27G E 1 22.7 6 A29GTW E 1.5 28.8 7 C 27G E 1.5 18.7 8 B 27G E 1.5 23.7

Example 10

Analysis of Pre-Filled Syringe Components Used in 27 G Thin Wall NeedleSyringes Filled with Anti-α4β7 Antibody Formulation

This study explored how various syringe manufacturers with a 27 G thinwall needle and various plunger (stopper) manufacturers and elastomericmaterials affected the mechanical properties of the pre-filled syringesystem and the stability of the formulation over time.

Three different syringe manufacturers and 4 different plunger (stopper)models were tested with 27 G ½%″ thin wall needles and a formulationcontaining 160 mg/mL protein, 125 mM arginine, 50 mM histidine, 25 mMcitrate, 0.2% PS80, at a pH of 6.5. All of the samples created andtested are shown in Table 29.

TABLE 29 Experimental details Plunger Run # Syringe (Stopper) 1 B F 2 BD 3 A D 4 B E 5 A F 6 C E 7 A E 8 C F 9 C D 10 C G

The samples were analyzed for appearance, injection force, SEC, CEX, andmicro-flow imaging at 5° C., 25° C., and 40° C. at various time pointsthat may have included 0, 1, 3, 6, and 12 months. The stability of theformulation as measured by SEC and CEX were similar to what wasdiscussed in Examples 1 and 2. For injection force testing, thebreakloose and glide force were measured. The results at the initialtime point are shown in Table 30.

TABLE 30 Initial Breakloose Breakloose Breakloose Plunger Glide InitialForce at 12 Force at 12 Force at 12 Run Syringe (Stopper) ForceBreakloose Months at Months at Months at # Manufacturer Type (N) Force(N) 5° C. (N) 25° C. (N) 40° C. (N) 1 B F 12.0 4.0 3.8 12.9 28.7 2 B D11.9 3.9 4.6 12.4 36.0 3 A D 7.0 4.0 6.5 5.1 6.4 4 B E 13.9 4.5 4.7 5.817.2 5 A F 5.7 4.1 3.0 17.5 23.9 6 C E 6.7 4.1 5.0 5.8 11.4 7 A E 7.97.6 4.3 10.4 6.1 8 C F 6.3 4.2 4.1 15.0 33.3 9 C D 5.9 4.8 3.9 4.4 10.010 C G 7.2 4.6 6.1 9.8 13.0

A statistical model showed that syringe manufacturers A and C weresimilar and had lower glide forces than manufacturer B, while plunger(stopper) E have slightly higher glide force than the other plunger(stoppers).

Generally, the initial breakloose forces were similar between all thesamples that were tested.

Over 12 months at 5° C., 25° C., and 40° C., the glide forces did notsignicantly change. However, the breakloose force for syringes withplunger (stopper) F increased by 12 months at 25° C. and 40° C.

Example 11

Analysis of Anti-α4β7 Antibody Formulation in Prefilled Syringes

This study determines how varying levels of protein concentration,polysorbate 80 concentration, citrate concentration, and pH affectsanti-α4β7 antibody formulations in a prefilled syringe format.

Part of the experimental design is created in JMP with a fractionfactorial of two levels of protein concentration (60 to 160 mg/mL), pH(6.0 to 6.3), polysorbate 80:protein molar ratio (0.723 to 1.5), andcitrate concentration (0 to 25 mM). These formulations have a constantvalue of Histidine concentration (50 mM) and Arginine (125 mM)(Formulations 1-8). Variations of these formulations with 25 mMHistidine are added (Formulations 9-10).

An additional set of formulations are developed to explore formulationswith no histidine present and only citrate acting as the buffer(Formulations 11-16). The levels of the inputs for all formulationsbeing explored are shown in Table 31. The constants used for allformulations are shown in Table 32.

TABLE 31 DOE variables and levels Nominal Values Variable Low HighProtein 60 160 Concentration (mg/mL) pH 6.0 6.3 PS80: Protein 0.723 1.5Molar Ratio Citrate 0 40 Concentration (mM) Histidine 0 50 Concentration(mM)

TABLE 32 Constants Constant Value Arginine Concentration (mM) 125Table 33 lists the formulations to be tested.

TABLE 33 Formulation details Antioxidant Formulation Protein Protein HisArg PS PS80:Protein Concentration # (mg/ml) (mM) (mM) (mM) 80% pH MolarRatio Antioxidant (mM) 1 60 0.400 50 125 0.038 6.3 0.723 Citric Acid 252 60 0.400 50 125 0.079 6.3 1.5 Citric Acid 0 3 157 1.047 50 125 0.2066.3 1.5 Citric Acid 25 4 160 1.067 50 125 0.101 6.3 0.723 Citric Acid 05 60 0.400 50 125 0.079 6.0 1.5 Citric Acid 25 6 110 0.733 50 125 0.0696.0 0.723 Citric Acid 0 7 160 1.067 50 125 0.101 6.0 0.723 Citric Acid25 8 160 1.067 50 125 0.210 6.0 1.5 Citric Acid 0  9* 160 1.067 25 1250.101 6.0 0.723 Citric Acid 25 10* 160 1.067 25 125 0.140 6.0 1 CitricAcid 25 11  160 1.067 0 125 0.101 6.3 0.723 Citric Acid 40 12  160 1.0670 125 0.210 6.3 1.5 Citric Acid 40 13  60 0.400 0 125 0.079 6.3 1.5Citric Acid 40 14* 160 1.067 0 125 0.210 6.1 1.5 Citric Acid 40 15* 1601.067 0 125 0.210 6.6 1.5 Citric Acid 40 16* 160 1.067 0 125 0.140 6.3 1Citric Acid 40

Each formulation is generated from a starting stock formulationcontaining anti-α4β7 antibody and diluted down with various excipientstock solutions. In order to achieve reasonable dilution volumes, theanti-α4β7 antibody stock solution used is shown in Table 34. Twodifferent TFF operations are performed to achieve the formulations TFF 1and 2. A portion of TFF 1 is used in a dialysis to achieve theformulation labeled “Dialysis”.

TABLE 34 Starting Formulation buffer details Total Total StartingProtein His Citrate Arg Formulation (mg/ml) (mM) (mM) (mM) pH TFF 1192.1 50 0 125 6.1 TFF 2 206.1 0 40 125 6.3 Dialysis 169.65 25 25 1256.0

For the dilution of the material to the desired formulation composition,stock solutions of each excipient in water are made at theconcentrations specified by Table 35.

TABLE 35 Stock solution details Excipient Concentration Histidine (mM)220 Arginine 625 Hydrochloride (mM) PS 80 (%) 2.5 Histidine 600Hydrochloride (mM) Citric Acid (mM) 1500 (pH 6.3) Citric Acid (mM) 1500(pH 6.0) Citrate (mM) 600 Sodium Citrate 800 (mM)

The dilution scheme for the formulations is detailed in Table 36 and 37.

TABLE 36 Dilution detail Starting Starting Formu- Formu- lation lationHis His*HCl Arg Citrate PS80 WFI (uL) (mg) (uL) (uL) (uL) Solution (uL)(uL) (uL) 1 4685.06 4961.01 1612.2 268.4 2063.0 250 227.3 5894.0 24685.06 4961.01 1612.2 268.4 2063.0 0 471.6 5899.7 3 12259.2 12981.31724.1 0.0 548.2 250.0 1234.0 0.0 4 12493.49 13229.36 696.7 0.0 501.3 0606.2 702.4 5 4685.06 4961.01 1002.8 491.9 2063.0 250 471.6 6035.7 68589.28 9095.18 545.0 334.4 1282.1 0 416.7 3832.4 7 12493.49 13229.3687.2 176.9 501.3 250 606.2 884.9 8 12493.49 13229.36 87.2 176.9 501.3 01257.6 483.5 9 12260.8 12941.30 38.2 16.8 147.8 12.3 525.3 0.0 1012260.8 12941.30 38.2 16.8 147.8 12.3 726.6 0.0

TABLE 37 Dilution details Starting Starting Citrate NaCitrate ArgFormulation (uL) Formulation (mg) (uL) (uL) (uL) PS80 (uL) WFI (uL) 119315.87 9944.69 8.3 128.0 536.8 484.9 1526.1 12 9315.87 9944.69 8.3128.0 536.8 1006.1 1005.0 13 3493.45 3729.26 30.5 402.5 1701.3 377.35995.0 14 5822.42 6215.43 22.0 67.4 335.5 628.8 623.9 15 5822.42 6215.430.0 300.0 335.5 628.8 413.3 16 5822.42 6215.43 5.2 80.0 335.5 419.2837.7

Compounding is performed based on the dilution scheme, and the startingformulation is weighed, while the other stock solutions are pipettedvolumetrically. Formulations are filtered. 0.5 mL of formulation isaliquotted into as many mL Long syringes as possible. The syringes arestoppered by the stoppering machine. The syringes are stored needledown.

Liquid formulations are tested analytically (appearance, pH, osmolality,density, DLS, SEC, CEX, and/or Brightwell) initially, and at 1 week, 40°C., 2 weeks, 40° C., month 25 and 40° C., months, and 25° C., months, 5and 25° C., 6 months, 5 and 25° C., 9 months, 5 and 25° C., and 12months, 5 and 25° C.

Specific formulation pulls according to Table 38 are also performed.

TABLE 38 Specific formulation pulls 1 2 1 2 3 6 9 12 FormulationTemperature Week Week Month Month Month Month Month Month Extras 1 5 — —— X X X X X 1 2 5 — — — X X X X X 1 3 5 — — — X X X X X 1 4 5 — — — X XX X X 1 5 5 — — — X X X X X 1 6 5 — — — X X X X X 1 7 5 — — — X X X X X0 8 5 — — — X X X X X 1 9 5 — — — — — — — — 3 10 5 — — — X X X — X 0 115 — — — — — — — — 5 12 5 — — — X — — — — 4 13 5 — — — — — — — — 5 14 5 —— — X — — — — 1 15 5 — — — — — — — — 2 16 5 — — — X — — — — 1 1 25 — — XX X X X X 1 2 25 — — X X X X X X 1 3 25 — — X X X X X X 1 4 25 — — X X XX X X 1 5 25 — — X X X X X X 1 6 25 — — X X X X X X 1 7 25 — — X X X X XX 1 8 25 — — X X X X X X 1 9 25 — — X — — — — — 3 10 25 — — X X — X — —0 11 25 — — X — — — — — 5 12 25 — — X X — — — — 4 13 25 — — X — — — — —6 14 25 — — X X — — — — 1 15 25 — — X — — — — — 2 16 25 — — X X — — — —1 1 40 X X X — — — — — 1 2 40 X X X — — — — — 1 3 40 X X X — — — — — 1 440 X X X — — — — — 1 5 40 X X X — — — — — 1 6 40 X X X — — — — — 1 7 40X X X — — — — — 1 8 40 X X X — — — — — 1 9 40 X X X — — — — — 0 10 40 XX X — — — — — 0 11 40 X X X — — — — — 0 12 40 X X X — — — — — 0 13 40 XX X — — — — — 0 14 40 X X X — — — — — 0 15 40 X X X — — — — — 0 16 40 XX X — — — — — 0

Example 12

A formulation containing 160 mg/mL protein, 50 mM histidine, 25 mMcitrate, 125 mM arginine at pH6.5 was tested for stability in either aglass syringe or two different COP plastic syringes. At 5° C. and 25° C.after 12 months, the amount of aggregates and monomer were comparablebetween the plastic and glass syringes.

TABLE 39 Amount Amount Change in Change in of of SEC SEC Monomer MonomerAggregates Aggregates After 12 After 12 After 12 After 12 Months MonthsFormulation PS80:Protein Syringe Months at Months at at 5° C. at 25° C.# Molar Ratio Material 5° C. (%) 25° C. (%) (%) (%) 1 1.5 COP 0.2 1.098.3 96.8 Manufacturer 1 2 1.5 COP 0.2 1.6 98.3 96.9 Manufacturer 2 31.5 Glass 0.2 1.4 98.4 96.8 4 1 Glass 0.2 1.6 98.3 96.8

Example 13: Bioavailability of Vedolizumab Administered by Subcutaneousand Intramuscular Injection

A phase I study of the bioavailability of vedolizumab administered bysubcutaneous and intramuscular injection to healthy male subjects wascompleted. A total of 42 healthy males were enrolled in the study. Thesubjects were divided into three groups (subcutaneous, intramuscular,and intravenous administration) of 14 subjects each. The subjects wereadministered 180 mg of vedolizumab on one day. The dose wasreconstituted from a lyophilized formulation of 60 mg/ml antibody in 50mM histidine, 125 mM arginine, 0.06% polysorbate 80, 10% sucrose, at pH6.3. For the intramuscular and subcutaneous subjects, the dose wasdivided into two injections of 1.5 ml each. Blood was sampled todetermine the plasma vedolizumab concentration and the bioavailabilityof vedolizumab in each set of subjects was determined.

No serious adverse events or significant infections, clinicallysignificant abnormalities, positive subjective/objective RAMPchecklists, nor clinically significant ECG findings were reported.

PK/PD modeling and simulation was completed to determine the dose andregimens of extra-vascular doses that result in similar exposures as tehintravenous doses in order to maintain this desired serum concentrationsat trough levels.

The absorption profile (FIG. 18) showed that concentrations of theintramuscular and subcutaneous doses generally overlap. There are noapparent gross differences in the absorption profiles of these routes ofadministration. The absolute bioavailability of vedolizumab following SCinjection was approximately 75% and following IM injection wasapproximately 80%.

Example 14. Modeling Subcutaneous Dose Regimens

PK/PD modeling and simulation was completed to determine the dose andregimens of extra-vascular doses that result in similar exposures as theintravenous doses in order to maintain certain serum concentrations attrough levels.

A final combined dataset (IV, SC and IM data) showed two compartmentlinear models parameterized in terms of clearance (CL) and centralvolume of distribution (V2), peripheral volume of distribution (V3), anextra-vascular route dependent absorptionrate constant (KA) and therelative bioavailability (compared to intravenous administration) of theextra-vascular doses (F). IIV terms were included on CL, V2 and V3 withbody weight as the only covariate influencing CL and V3 through anallometric effect.

Model acceptability and predictability was demonstrated throughbootstrap parameter estimates, visual predictive checks and goodness offit plot. Analysis of the model identified body weight as a predictorfor the PK of vedolizumab with the variability in the PK attributed tobetween subject and within subject components.

Once the model was demonstrated to be adequate for simulation,simulations were performed in order to assess the effect of route ofadministration (IV, IM or SC), and assess the effect of frequency ofdosing (weekly, every 2 weeks, every 4 weeks, and every 8 weeks) on thesteady state trough concentrations. Based on these values and therelative bioavailability of vedolizumab following IM and SCadministration (F=69.5%), doses were selected to achieve similar troughconcentrations as the IV doses.

Simulations modeled doses and regimens to match intravenous inductionand maintenance regimens. The targets were both exposure (area underserum drug concentration-time curve (AUC)) and trough drugconcentration. Tables 40-43 provide results of simulations.

TABLE 40 Induction regimen matching an IV AUC during weeks 0-6 RouteDose Frequency IV 300 mg Week 0 & 2 SC 485 mg Week 0 & 2 SC 160 mg Everyother day (6 doses) SC >160 mg Weekly (6 doses)

TABLE 41 Induction regimen matching an IV trough concentration, weeks0-6 Route Dose Frequency IV 300 mg Week 0 & 2 SC >160 mg Week 0 & 2 SC100 mg Every week (6 doses) SC 160 mg Every other day (for 2 weeks)

TABLE 42 Maintenance regimen matching a 300 mg IV dose every 4 weeksDose matching Dose 4wk IV steady matching state trough 4wk IV FrequencyRoute concentration AUC Once every IV 300 300 4 weeks IM 432 432 SC 432432 Once every IV 115 150 2 weeks IM 165 216 SC 165 216 Every week IV 5075 IM 72 108 SC 72 108

TABLE 43 Maintenance regimen matching a 300 mg IV dose every 8 weeksDose matching Dose 8 wk IV steady matching state trough 8 wk FrequencyRoute concentration IV AUC Once every 8 IV 300 300 weeks IM 432 432 SC432 432 Once every 4 IV  90 150 weeks IM 125 216 SC 125 216 Once every 2IV  35  75 weeks IM  50 108 SC  50 108 Every week IV  15   37.5 IM  22 54 SC  22  54

Example 15: Phase 2a Multiple Dose Study

A Phase 2a multiple dose study can assess the safety, tolerability andsteady state PK of vedolizumab following multiple doses of vedolizumabby the subcutaneous administration route and to assess the relativebioavailability of the subcutaneous regimen compared with theintravenous regimen. The development of HAHA and neutralizing HAHA andthe effect on PD of multiple doses of vedolizumab following subcutaneousadministration can be assessed.

Ulcerative colitis patients having a partial Mayo score of 1-12 andCrohn's disease patients having a CDAI greater than 150 can be includedin the study. Cohorts can receive an induction regimen of vedolizumab(300 mg) administered IV at weeks 0 and 2, followed by a maintenanceregimen of either

Vedolizumab (300 mg) administered IV every 4 weeks at weeks 6-22

Vedolizumab (300 mg) administered IV every 8 weeks at weeks 6-22

Vedolizumab (108 mg) administered SC every week at weeks 6-22

Vedolizumab (108 mg) administered SC every 2 weeks at weeks 6-22

Vedolizumab (165 mg) administered SC every 3 weeks at weeks 6-22.

Samples can be collected before dosing on day 1, and then again on day 1(12 hours), 2, 3, 5, 8, 15, 29, 43, 127, 127 (12 hours), 128, 129, 131,134, 141, and 155 to assess PK and PD.

Example 16: Long-Term Clinical Experience with Vedolizumab for theTreatment of IBD

A phase 2 open-label safety extension study was completed to assess thelong-term pharmacokinetics (PK), pharmacodynamics (PD), safety, andefficacy of vedolizumab. Patients were aged 18 to 75 years old, and hadeither previously participated in an earlier PK/PD/safety study inulcerative colitis patients or had IBD symptoms for at least 2 monthsconfirmed endoscopically and/or histopathologically and/orradiologically within 36 months of screening.

All patients received an intravenous dosing regimen of either 2 mg/kg or6 mg/kg of vedolizumab (5 mg/mL antibody, 20 mM citrate/citric acid, 125mM sodium chloride, 0.05% polysorbate 80, pH 6.0 (stored long term −70°C. and up to 3 mo −20° C.)) on days 1, 15 and 43, followed by a doseevery 8 weeks for up to a total of 78 weeks. Patients were eithertreatment-naïve ulcerative colitis or Crohn's disease patients, orulcerative colitis patients that had participated in an earlier clinicaltrial.

Efficacy/quality of life (QoL); partial Mayo score (PMS), Crohn'sdisease activity index (CDAI), and Inflammatory Bowel DiseaseQuestionnaire (IBDQ) were used to assess the results of the study.

PK Results

Mean pre-infusion vedolizumab concentrations were dose proportional, andremained steady and detectable throughout the study.

PD Results

Receptors (% ACT-1+[CD4+CD45RO HIGH] and % MADCAM+[CD4+CD45RO HIGH] werealmost fully inhibited throughout the study period at all dose levels.

Partial Mayo Score

Baseline mean PMS was higher for treatment-naïve ulcerative colitispatients (5.4) than for ulcerative colitis rollover patients (2.3). Byday 43, mean PMS showed a pronounced decrease for both rollover andtreatment-naïve ulcerative colitis patients. By day 155, mean scores ofthe two groups were similar. Mean PMS continued to decrease through day267, and leveled off thereafter.

Crohn's Disease Activity Index

CD patients' mean CDAI decreased from 294.6 at baseline to 237.7 at Day43, and continued to decrease through day 155 (156.1).

IBDQ

Ulcerative colitis rollover patients had the highest mean IBDQ scores atbaseline. By day 43, mean IBDQ scores had increased in all three diseasegroups. Mean IBDQ scores continued to increase over time in all 3disease groups, reaching a maximum at day 155 for Crohn's Diseasepatients, and at day 491 for treatment-naïve ulcerative colitis patientsand ulcerative colitis rollover patients.

C-Reactive Protein

Both ulcerative colitis rollover and Crohn's disease patients showeddecreased mean CRP levels through day 155 and then leveled off.Treatment-naïve ulcerative colitis patients had a lower mean CRP levelat baseline than ulcerative colitis rollover patients (2.28 v. 7.09).Mean CRP levels of the treatment-naïve ulcerative colitis patientsremained relatively constant at all time points assessed.

Other Safety Results

No systematic opportunistic infections (including PML) were reportedduring the study. One patient tested positive for JC viremia at a singletime point, though was negative for JCV at all other time points. Threeof 72 patients (4%) had positive HAHA results (two of these weretransiently positive). The study showed no evidence of liver toxicity,lymphocytosis, or lymphopenia, or any other drug-associated laboratorychanges.

CONCLUSIONS

Vedolizumab administered at 2.0 or 6.0 mg/kg once every 8 weeks for upto 78 weeks achieved target receptor saturations, was associated withdurable mean decreases in disease activity and improved IBDQ scores, wasgenerally safe and well tolerated, and demonstrated acceptableimmunogenicity.

Example 17: Induction and Maintenance of Response and Remission in

Patients with Moderately to Severely Active Ulcerative Colitis A singletrial comprising two randomized, double blind, multi-center studiesdesigned to evaluate induction and maintenance of response and remissionin patients with moderately to severely active ulcerative colitis.Demographic and baseline disease characteristics were comparable acrossall treatment groups.

The induction study, using intravenous administration, compared placeboagainst vedolizumab, at a 300 mg dose reconstituted from a lyophilizedformulation of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine,0.06% polysorbate 80, 10% sucrose, at pH 6.3, with an endpoint at 6weeks after 2 doses of vedolizumab.

The maintenance study, using the same formulation and route ofadministration as the induction study, compared placebo againstvedolizumab dosed every four weeks, and placebo against vedolizumabdosed every eight weeks. The endpoint of this study was at 52 weeks,analyzing the induction responder population.

Blood samples were collected to measure concentrations of vedolizumabduring the study. The mean serum concentration of vedolizumab at the endof the induction phase was 20 to 30 μg/mL. The mean vedolizimab troughserum concentrations at steady state after 30 min IV infusion of 300 mgdose administration were between 9 to 13 μg/mL for the q8wks regimen (8week regimen) and between 35 to 40 μg/mL for the q4wks regimen (4 weekregimen). At the end of infusion, the vedolizimab median plasmaconcentrations were between 98 and 101 μg/mL for the q8ks regimen andaround 129 and 137 μg/mL for the q4 wks regimen.

Summaries of the responses of the induction and maintenance studies areprovided in Tables 44-47. A significantly greater proportion ofvedolizumab-treated patients achieved clinical response, remission, andmucosal healing at 6 weeks, compared with placebo (Table 44). 39% of theinduction phase intent-to-treat population had prior anti-TNFα failure.Clinical response and remission rates were higher in vedolizumab thanplacebo patients among both those with prior anti-TNF failure and thosewith no prior anti-TNF exposure. In preliminary analyses through week 6,rates of adverse events (AEs), serious AEs, and adverse events leadingto study discontinuation were higher in the placebo group thanvedolizumab group. A significantly greater proportion of vedolizumabpatients than placebo patients achieved clinical remission, mucosalhealing, and corticosteroid-free remission at 52 wks and durableresponse and remission (Table 45). 32% of the maintenance studypopulation had prior anti-TNFα failure. Clinical remission and durableclinical response rates were greater with vedolizumab than placebo inboth TNF failure and TNF naïve patients. In the safety population(N=895) for wks 0-52, rates of adverse events (AEs), serious AEs, andserious infections were similar between vedolizumab and placebo groups.No increase in rates of opportunistic or enteric infections was observedin the vedolizumab group.

TABLE 44 Induction Study Results-Primary and Key Secondary EndpointsEfficacy Endpoints Placebo Vedolizumab Difference/RR P value Clinical25.5% 47.1% 21.7%/1.8 <0.0001 Response (%) Clinical 5.4% 16.9% 11.5%/3.10.0010 Remission (%) Mucosal 24.8% 40.9 16.1%/1.6 0.0013 Healing (%)

TABLE 45 Maintenance Study Results-Primary and Key Secondary EndpointsDifference/RR Placebo VDZ Q8 VDZ Q4 Q8 vs. Pb Efficacy Endpoint N = 126N = 122 N = 125 Q4 vs. Pb P value Clinical Remission 15.9  41.8 44.826.1/2.7 <0.0001 (%) 29.1/2.8 <0.0001 Durable Response 23.8  56.6 52.032.8/2.4 <0.0001 (%) 28.5/2.2 <0.0001 Mucosal Healing (%) 19.8  51.656.0 32.0/2.6 <0.0001 36.3/2.8 <0.0001 Durable Remission 8.7 20.5 24.011.8/2.4  0.0090 (%) 15.3/2.8  0.0011 Corticosteroid-free 13.9  31.445.2 17.6/2.3  0.0133 Remission (%) n = 72 n = 70 N = 73 31.4/3.3<0.0001

TABLE 46 Induction Study: Clinical Response and Remission at 6 Weeks inPatients with Prior Anti-TNF-α Antagonist Failure and Without Anti-TNFExposure, ITT Population Patients with Prior Anti-TNF-α AntagonistFailure (39%) Endpoint Placebo Vedolizumab Difference 95% Cl N = 63 N =82 Clinical 20.6  39.0  18.4   3.9, 32.9 Response (%) Clinical 3.2 9.86.6 −9.8, 22.8 Remission (%) Patients Without Anti-TNF-α AntagonistExposure (55%) Placebo Vedolizumab Difference 95% Cl N = 76 N = 130Clinical 26.3  53.1  26.8  13.7, 39.9 Response (%) Clinical 6.6 23.1 16.5   2.4, 30.2 Remission (%)

TABLE 47 Clinical Remission and Durable Clinical Response at 52 Weeks:Patients with Prior Anti-TNF-α Antagonist Failure or Without Anti-TNF-αAntagonist Exposure ITT Population Patients with Prior Anti-TNF-αAntagonist Failure (32%) Endpoint Placebo VDZ VDZ Difference 95% Cl N =38 Q8 Wks Q4 Wks Q8 wks vs N = 43 N = 40 Placebo Q4 wks vs. PlaceboClinical remission 5.3 37.2 35.0 31.9 10.3, 51.4 (%) 29.7  7.4, 49.4Durable Clinical 15.8  46.5 42.5 30.7 11.8, 49.6 Response (%) 26.7  7.5,45.9 Patients without Anti-TNF-α Antagonist Exposure (60%) Placebo VDZVDZ Difference 95% Cl N = 79 Q8 wks Q4 wks Q8 wks vs. N = 72 N = 73Placebo Q4 wks vs. Placebo Clinical Remission 19.0  45.8 47.9 26.8 12.4,41.2 (%) 29.0 14.6, 43.3 Durable Clinical 26.6  65.3 56.2 38.7 24.0,53.4 Response (%) 29.6 14.6, 44.6

Example 18: Induction and Maintenance of Response and Remission inPatients with Moderately to Severely Active Crohn's Disease

A single trial comprising two randomized, double blind, multi-centerstudies designed to evaluate induction and maintenance of response andremission in patients with moderately to severely active Crohn'sDisease. Demographic and baseline disease characteristics werecomparable across all treatment groups.

The induction study, using intravenous administration, compared placeboagainst vedolizumab, at a 300 mg dose reconstituted from a lyophilizedformulation of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine,0.06% polysorbate 80, 10% sucrose, at pH 6.3, with an endpoint at 6weeks after 2 doses of vedolizumab.

The maintenance study, using the same formulation and route ofadministration as the induction study, compared placebo againstvedolizumab dosed every four weeks, and placebo against vedolizumabdosed every eight weeks. The endpoint of this study was at 52 weeks,analyzing the induction responder population.

Surprisingly, this study showed that Q4 and Q8 week groups yielded verysimilar results. Summaries of the responses of the induction andmaintenance studies are provided in Tables 48-51. A significantlygreater proportion of vedolizumab-treated patients achieved clinicalremission and enhanced response, compared with placebo (Table 48).Clinical remission and enhanced response rates were higher invedolizumab than placebo patients among both those with prior anti-TNFfailure and those with no prior anti-TNF exposure. Rates of adverseevents (AEs), serious AEs, and serious infections were similar betweenvedolizumab and placebo groups. No increase in rates of opportunistic orenteric infections was observed in the vedolizumab group.

TABLE 48 Induction Study Results-Primary and Secondary Endpoints PlaceboVedolizumab Adjusted Endpoints N = 148 N = 220 Difference/RR P valueClinical 6.8% 14.5% 7.8%/2.1 0.0206 Remission (%) Enhanced 25.7% 31.4%5.7%/1.2 0.2322 Response (%) Mean CRP −3.6 −2.9 0.9288 Change N = 147 N= 220 (μg/mL)

TABLE 49 Maintenance Study Results-Primary and Key Secondary EndpointsAdj. Difference/RR Efficacy Placebo VDZ Q8 VDZ Q4 Q8 vs. Pb P Endpoint N= 153 N = 154 N = 154 Q4 vs. Pb value Clinical Remission 21.6 39.0 36.417.4/1.8  0.0007 (%) 14.7/1.7  0.0042 Enhanced Response 30.1 43.5 45.513.4/1.4  0.0132 (%) 15.3/1.5  0.0053 Corticosteroid-free 15.9 31.7 28.815.9/2.0  0.0154 Remission (%) N = 82 N = 82 N = 80 12.9/1.8  0.0450Durable Remission 14.4 21.4 16.2 7.2/1.5 0.1036 (%) 2.0/1.1 0.6413

TABLE 50 Clinical Remission and Enhanced Response at 6 Weeks in Patientswith Prior Anti-TNF-α Antagonist Failure and Without Anti-TNF Exposure,ITT Population Patients with Prior Anti-TNF-α Antagonist Failure (48%)Endpoint Placebo Vedolizumab Difference 95% Cl N = 70 N = 105 Clinical4.3 10.5 6.2  (−9.1, 21.3) Remission (%) Enhanced 22.9  23.8 1.0 (−11.8,13.7) Response (%) Patients Without Anti-TNF-α Antagonist Exposure (50%)Placebo Vedolizumab Difference 95% Cl N = 76 N = 130109 Clinical 9.217.4 8.2  (−1.4, 17.9) Remission (%) Enhanced 30.3  42.2 11.9   (−1.9,25.8) Response (%)

TABLE 51 Clinical Remission and Enhanced Response at 52 Weeks: Patientswith Prior Anti-TNF-α Antagonist Failure or Without Anti-TNF-αAntagonist Exposure ITT Population Patients with Prior Anti-TNF-αAntagonist Failure (51%) Endpoint Placebo VDZ VDZ Difference 95% Cl N =78 Q8 Wks Q4 Wks Q8 wks vs N = 82 N = 77 Placebo Q4 wks vs. PlaceboClinical 12.8 28.0 27.3 15.2  (3.0, 27.5) remission (%) 14.5  (2.0,26.9) Enhanced 20.5 29.3 37.7 8.8 (−4.6, 22.1)  Response (%) 17.1  (3.1,31.2) Patients without Anti-TNF-α Antagonist Exposure (45%) Placebo VDZVDZ Difference 95% Cl N =71 Q8 wks Q4 wks Q8 wks vs N = 66 N = 71Placebo Q4 wks vs. Placebo Clinical 26.8 51.1 46.5 24.8  (8.9, 40.6)remission (%) 19.7  (4.2, 35.2) Enhanced 38.0 60.6 53.5 22.6  (6.3,38.9) Response (%) 15.5  (−0.7, 31.7) 

Example 19: Induction of Response and Remission in Patients withModerate to Severely Active Crohn's Disease

A randomized, double blind, placebo controlled multi-center study wascompleted to evaluate the induction effect of vedolizumab at 300 mgdoses (reconstituted from a formulation of 60 mg/ml antibody in 50 mMhistidine, 125 mM arginine, 0.06% polysorbate 80, 10% sucrose, at pH6.3which was lyophilized), in TNFα antagonist failure patients at week 6(after 2 doses—0 and 2 weeks) and at week 10 (after 3 doses). The studyconsisted of 416 patients, 75% of whom were TNFα antagonist failures,and 25% of whom were TNFα naïve. Demographics and concomitant IBDmedication were balanced across treatment groups. Baseline diseasecharacteristics were also balanced across treatment groups, except forbaseline disease activity.

The primary endpoint designated for the study was week 6 remission (%)in anti-TNF-α antagonist failure population. The key secondary endpointsthat were evaluated (sequential testing procedure) were: week 6remission (%) in overall population, week 10 remission (%) in anti-TNF-αantagonist failure and overall population (using Hochberg procedure),week 6 and 10 sustained remission (%) in anti-TNF-α antagonist failureand overall population (using Hochberg procedure), and week 6 enhancedresponse (%) in anti-TNF-α antagonist failure population.

TABLE 52 Baseline CDAI: Placebo Vedolizumab p-value TNF ITT: Mean 306.1(55.43) 316.1 (52.63) 0.0945 (Std Dev) Overall ITT: Mean 301.3 (54.97)313.9 (53.17) 0.0153 (Std Dev)

TABLE 53 Induction Study Results: Primary and Key Secondary EndpointsTNF ITT (N = 315) Overall ITT (N = 416) PLA VDZ Diff P- PLA VDZ DiffEndpoints N = 157 V = 158 (RR) value N = 207 N = 209 (RR) P-valuePrimary 12.1% 15.2%  3.0% 0.4332 Wk6 (1.2) Remission 1st 12.1% 19.1% 6.9% 0.0478 Secondary (1.6) Wk6 Remission 2nd 12.1% 26.6% 14.4% 0.0012  13% 28.7% 15.5% <0.0001 Secondary (2.2) (2.2) Wk10 Remission Sustained 8.3% 12.0%  3.7% 0.2755  8.2% 15.3%   7% 0.0249 Remission (1.4) (1.9)(both Wk 6&10) Enhanced 22.3% 39.2% 16.9% 0.0011 Response (1.8)(CDAI100)

TABLE 54 Results in Anti-TNF-α Antagonist Naïve Patients (n = 101, 24%of overall) Placebo Vedolizumab Difference 95% % % % Cl Remission Week 612 31.4 19.1 (3.3, 35.0) Remission Week 10 16 35.3 19.2 (2.4, 35.8)

TABLE 55 Study Results: Clinical Remission at Weeks 6 and 10, KeySubgroup-Previous Tx Failures, ITT Overall Subgroup Variable Placebo VDZDiff 95% Cl Any prior anti- N 156 155 TNF failure Wk 6 12.8 14.8 2(−5.7, 9.7)  (75% of ITT) Rem (%) Wk 10 12.8 26.5 13.6  (4.9, 22.3) Rem(%) Prior N 45 44 immunomodulator Wk 6 11.1 31.8 20.7 (−0.5, 39.7)failure but not Rem (%) anti-TNF failure Wk 10 15.6 31.8 16.3 (−1.1,33.6) (21% ITT) Rem (%) Prior N 5 9 corticosteroid Wk 6 0 33.3 33.3(−23.9, 75.7)  failure only Rem (%) (3% ITT) Wk 10 0 44.4 44.4 (−13.4,85.3)  Rem (%)

The study showed that TNF-α antagonist failure patients required 3 dosesfor induction of remission. Remission rates in TNF-α antagonist failurepatients increased between week 6 and week 10, but only for thevedolizumab group (not placebo). Remission rates for TNF-α antagonistnaïve patients did not increase substantially between week 6 and 10. Ofthe TNF-α antagonist failure population with a high degree of diseaseseverity, 43% never responded to a TNF-α antagonist, and 45% lostresponse.

Example 20: Stability

Various different anti-α4β7 antibody formulations were tested forstability over the course of 6 to 24 months at 5° C. (Tables 6 and 7).Formulations having a pH of 6.0-6.2 showed approximately less than 4%major species degradation after 6 months and at 24 months.

Various different anti-α4β7 antibody formulations were tested forstability by SEC for up to 24 months (Tables 4 and 5). The formulationswith 60 mg/mL protein concentration and containing 25 mM citrate had achange in aggregates of 0.1-0.2% after 2 years, while formulationscontaining 160 mg/mL protein and 25 mM citrate had an increase ofaggregates of approximately 0.3% over 2 years. There was an increase of0.6-1.1% aggregates for formulations containing 60, 110, or 160 mg/mLprotein with no citrate. For the formulations tested containing citrate,but no histidine, after 12 months and 24 months, there was approximately0.3-0.4% growth of aggregates.

Example 21: Determination of the Effect of Vedolizumab on the CD4:CD8Ratio

Healthy subjects ages 18-45 were treated with a single 450 mg dose ofvedolizumab reconstituted from a lyophilized formulation of 10% sucroseand diluted into an infusion system of 0.9% saline. Cerebrospinal fluid(CSF) was collected by lumbar puncture before (baseline) and 5 weeksafter the single 450-mg dose of vedolizumab. Each subject served ashis/her own control.

A 5-week time point was selected based on a previous study that showedpatients with MS treated with natalizumab demonstrated effects on CSFCD4+:CD8+ lymphocyte ratio and reduction in number of brain lesionsafter only one dose (Stuve et al. Arch Neurol. 2006; 63:1383-1387; Stuveet al. Ann Neurol. 2006; 59:743-747. Miller et al. N Engl J Med. 2003;348(1):15-23); and also because at 5 weeks, a 450-mg dose of vedolizumabis sufficient to saturate the target and provides serum concentrationsthat exceed estimated steady-state trough levels associated with thephase 3 dose regimen of 300 mg every 4 weeks.

Approximately 15 mL CSF was obtained from each subject forimmunophenotyping. CSF samples were included for analyses if they metthe following criteria: ≤10 RBCs/μL per sample (to minimize peripheralblood contamination); negative CSF culture result; adequate T-lymphocytenumbers in each flow cytometry sample; and no detection of serumantibodies to vedolizumab.

Week 5 median (34.80 μg/mL) and individual subject serum vedolizumabconcentrations (range 24.9-47.9 μg/mL) were higher than projectedsteady-state trough concentration (˜24 μg/mL) for the phase 3 doseregimen. A high degree (>90%) of α4β7 receptor saturation was observedat week 5 as measured by MAdCAM-1-Fc, indicating vedolizumab'ssaturation of its target at the time of endpoint assessment.

Vedolizumab was not detected in any CSF sample (detection limit=0.125μg/mL).

Effect on CD4+ and CD8+T Lymphocyte Numbers and Ratio

Vedolizumab did not significantly reduce CD4+:CD8+ ratio (Table 56).None of the subjects had a postdose CD4+:CD8+ ratio <1 (p<0.0001(1-sided t-test)). Vedolizumab did not significantly reduce the numberof CD4+ or CD8+T lymphocytes in CSF. In addition, there were nosignificant changes in CSF % CD4+ and % CD8+T lymphocytes (Table 57).Also, no significant changes in peripheral blood WBC, CD4+ and CD8+memory T lymphocytes (Table 58) were observed.

TABLE 56 Effect of Treatment on CSF CD4+: CD8+ Ratio (EvaluablePopulation, n = 13) CD4+: CD8+ Ratio Baseline Week 5 Difference† CD4+:CD8+ ratio 3.59 (0.273) 3.60 (0.265)* 0.01 (0.197) Mean (SE) Range1.53-5.67 1.42-5.15 90% 2-sided CI for 3.00-4.19 3.132, 4.077 ratio 90%2-sided CI for −0.337, 0.363 difference CI = confidence interval*p<0.0001 (one sided one sample t-test for H0: μ<1 vs H1: μ>=1).†Difference is defined as week 5 ratio minus baseline ratio

TABLE 57 Treatment Effect on CSF CD4+ and CD8+ Lymphocyte Count(Evaluable Population, n = 13) Baseline Week 5 CD4+ as % of 75.160(7.3831) 74.215 (6.3732) Lymphocytes, mean (SD) CD8+ as % of 22.272(5.4320) 22.007 (6.1624) Lymphocytes, mean (SD)

TABLE 58 Peripheral Blood Memory T Lymphocytes (RO+) Counts (EvaluablePopulation, n = 13) Baseline Week 5 Mean (SD) Mean (SD) CD4+CD45RO+27.85 (4.98) 27.06 (5.02) CD8+CD45RO+(%) 11.24 (3.40) 10.78 (2.98)

SUMMARY

Vedolizumab did not affect CSF CD4+ and CD8+ cell counts or CD4+:CD8+ratio in healthy volunteers after a single 450 mg dose. None of thesubjects had a reduction in the post-dose CSF CD4+:CD8+ ratio to lessthan 1. Vedolizumab was not detected in CSF. In addition, there was nochange observed in the total WBCs or memory T lymphocyte CD4+ and CD8+subsets in peripheral blood. Saturation of the target (α4β7) in bloodoccurred in all subjects at the time of endpoint assessment. The CSFCD4+ and CD8+ lymphocyte levels and ratio were similar to thosepreviously reported in the literature.

These results are consistent with vedolizumab's lack of effect on bothphysiologic CNS immune surveillance and pathologic CNS inflammation ofmonkeys.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

TABLE 59 Sequences SEQ ID NO: Sequence Shown Description  1 FIG. 1DNA encoding heavy chain of  humanized anti-α4β7 immunoglobulin  2FIG. 1 Amino acid sequence of heavy chain of humanized anti-α4β7immunoglobulin  3 FIG. 2 DNA encoding the light chain of humanized anti-α4β7 immunoglobulin  4 FIG. 2 Amino acid sequence of light chainof humanized anti- α4β7 immunoglobulin  5 FIG. 3 Mature humanizedlight chain of LDP-02  6 FIG. 4 Generic human kappa light chainconstant region  7 FIG. 4 Generic murine kappa light chainconstant region  8 Referenced on page 39 CDR1 of heavy chain SYWMHmouse ACT-1 antibody (SEQ ID NO: 8)  9 Referenced on page 39CDR2 of heavy chain EIDPSESNTNYNQKFKG mouse ACT-1 antibody(SEQ ID NO: 9) 10 Referenced on page 39 CDR3 of heavy chain GGYDGWDYAIDYmouse ACT-1 antibody (SEQ ID NO: 10) 11 Referenced on page 39CDR1 of light chain RSSQSLAKSYGNTYLS mouse ACT-1 antibody(SEQ ID NO: 11) 12 Referenced on page 39 CDR2 of light chain GISNRFSmouse ACT-1 antibody (SEQ ID NO: 12) 13 Referenced on page 39CDR3 of light chain LQGTHQPYT mouse ACT-1 antibody (SEQ ID NO: 13) 14FIG. 7 human GM607 CL antibody kappa light chain variable region 15FIG. 7 Human 21/28 CL antibody heavy chain variable region

1. A stable liquid pharmaceutical formulation comprising a mixture of ahumanized anti-α4β7 antibody, an antioxidant or chelator, and at leastone free amino acid, wherein the anti-α4β7 antibody comprises a lightchain variable region comprising a CDR1 comprising SEQ ID NO:11, a CDR2comprising SEQ ID NO: 12, and a CDR3 comprising SEQ ID NO: 13, andcomprises a heavy chain variable region comprising a CDR1 comprising SEQID NO:8, a CDR2 comprising SEQ ID NO: 9, and a CDR3 comprising SEQ IDNO:
 10. 2. The stable liquid pharmaceutical formulation of claim 1,wherein the molar ratio of the anti-α4β7 antibody to the antioxidant orchelator is about 1:4 to about 1:100.
 3. (canceled)
 4. (canceled)
 5. Thestable liquid pharmaceutical formulation of claim 1, wherein saidantioxidant or chelator is citrate.
 6. (canceled)
 7. The stable liquidpharmaceutical formulation of claim 1, wherein said free amino acid isselected from the group consisting of histidine, alanine, arginine,glycine, glutamic acid and combinations thereof.
 8. The stable liquidpharmaceutical formulation of claim 1, wherein said formulation furthercomprises a surfactant.
 9. The stable liquid pharmaceutical formulationof claim 8, wherein the molar ratio of the antioxidant or chelator tothe surfactant is about 3:1 to about 156:1. 10-12. (canceled)
 13. Thestable liquid pharmaceutical formulation of claim 1, wherein saidformulation comprises at least about 60 mg/ml to about 160 mg/mlanti-α4β7 antibody. 14-21. (canceled)
 22. The stable liquidpharmaceutical formulation of claim 1, wherein said antibody isvedolizumab. 23-28. (canceled)
 29. A method for treating a human patientsuffering from inflammatory bowel disease, wherein the method comprisesthe step of: administering to a patient suffering from inflammatorybowel disease, a humanized immunoglobulin or antigen-binding fragmentthereof having binding specificity for human α4β7 integrin, wherein thehumanized immunoglobulin or antigen-binding fragment thereof isadministered to the patient according to the following dosing regimen:(a) initial doses of 165 mg of the humanized immunoglobulin orantigen-binding fragment thereof as a subcutaneous injection every otherday for six doses; (b) followed by a seventh and subsequent doses of 165mg of the humanized immunoglobulin or antigen-binding fragment thereofas a subcutaneous injection every two weeks or every four weeks asneeded; wherein the dosing regimen induces a clinical response andclinical remission in the inflammatory bowel disease of the patient;further wherein the humanized immunoglobulin or antigen-binding fragmentcomprises an antigen binding region of nonhuman origin and at least aportion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment has binding specificity forthe α4β7 complex, wherein the antigen-binding region comprises the CDRs:Light chain: CDR1 SEQ ID NO:9 CDR2 SEQ ID NO:10 CDR3 SEQ ID NO:11 Heavychain: CDR1 SEQ ID NO:12 CDR2 SEQ ID NO:13 CDR3 SEQ ID NO:14.
 30. Themethod of claim 29, wherein the patient had a lack of an adequateresponse with, loss response to, or was intolerant to treatment with atleast one of an immunomodulator, a tumor necrosis factor-alphaantagonist or combinations thereof.
 31. The method of claim 29, whereininflammatory bowel disease is Crohn's disease or ulcerative colitis. 32.(canceled)
 33. (canceled)
 34. The method of claim 29, wherein the dosingregimen results in mucosal healing in patients suffering from moderateto severely active ulcerative colitis.
 35. The method of claim 29,wherein the dosing regimen results in a reduction, elimination orreduction and elimination of corticosteroids use by the patient.
 36. Themethod of claim 29, where the patient previously received treatment withat least one corticosteroid for the inflammatory bowel disease. 37-63.(canceled)
 64. The method of claim 29, wherein the anti-α4β7 antibody isvedolizumab.
 65. A method of preparing a pharmaceutical formulationcomprising an anti-α4β7 antibody, said method comprising diafiltering afirst solution comprising a first concentration of the anti-α4β7antibody in a tangential flow filtration system to obtain a secondsolution, adding polysorbate to the second solution, and diluting thesecond solution to obtain a third solution comprising a concentration ofthe anti-α4β7 antibody that is lower than the first concentration,thereby preparing a pharmaceutical formulation comprising an anti-α4β7antibody, wherein the anti-α4β7 antibody comprises a light chainvariable region comprising a complementarity determining region 1 (CDR1)comprising SEQ ID NO:11, a CDR2 comprising SEQ ID NO:12, and a CDR3comprising SEQ ID NO:13, and comprises a heavy chain variable regioncomprising a CDR1 comprising SEQ ID NO:8, a CDR2 comprising SEQ ID NO:9,and a CDR3 comprising SEQ ID NO:10.
 66. The method of claim 65, whereinthe third solution comprises an antibody concentration of about 160mg/ml.
 67. The method of claim 65, wherein the polysorbate ispolysorbate
 80. 68. The method of claim 65, wherein the second solutioncomprises citrate, histidine, and arginine.
 69. The method of claim 65,wherein the anti-α4β7 antibody is vedolizumab.